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Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Purpose Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system. Methods Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates. Results Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus. Conclusions These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.

Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina paired-end, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The final genome assembly is 177 Mbp with contig N50 size of 75 kbp and a scaffold N50 size of 2.3 Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts. Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.10283018

The effect of Toxocara canis antigens on cytokine production by human THP-1 macrophages was studied in vitro. Toxocara Excretory-Secretory products (TES) and recombinant mucins (Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5) as well as deglycosylated forms of these antigens were used in the study. TES products stimulated macrophages to produce the innate proinflammatory IL-1β, IL-6, and TNF-α cytokines regardless of the presence of glycans. Recombinant mucins induced glycan-dependent cytokine production. Sugar moieties led to at least 3-fold higher production of regulatory IL-10 as well as proinflammatory cytokines. The presence of glycans on mucins also affected the downstream signalling pathways in stimulated cells. The most prominent difference was noted in AKT and AMPK kinase activation. AKT phosphorylation was observed in cells stimulated with glycosylated mucins, whereas treatment with deglycosylated antigens led to AMPK phosphorylation. MAP kinase family members such as JNK and p38 and c-Jun transcription factor were phosphorylated in both cases what suggests that toll-like receptor signalling may be involved in mucin-treated macrophages. This pathway is however modified by other signalling molecules as only mucins containing intact sugars significantly induced the production of cytokines.

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets.

14-3-3 proteins are present in all eukaryotic organisms and are ubiquitously expressed in a broad range of tissues and cellular compartments. They are regulatory adapter proteins that play key roles in a variety of signaling pathways, and have been proposed as suitable targets for the control and detection of certain parasites. Trichinella britovi is a widely-distributed parasitic nematode, transmitted through ingestion of meat products containing invasive larvae. The present study describes the cloning and expression of Tb14-3-3, and investigates the immunological and protective potential of the recombinant protein. Immunization of mice with rTb14-3-3 triggered an IgG response, and significant differences, in the profiles of secreted cytokines observed in vitro, between experimental groups. Nonetheless, neither specific antibodies, nor increased secretion of IFN&gamma, IL-4, and IL-10 cytokines, conferred greater protection against infection. No reduction in larval burden was observed during recovery at 48 dpi. Additionally, rTb14-3-3 was not recognized by sera from the infected control mice, except for one, suggesting some mismatch between native and recombinant Tb14-3-3 antigenic sites. Therefore, before 14-3-3 can be considered a potential tool for Trichinella detection and vaccination, more research regarding its target proteins, and actual specific function, is needed.

No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock.

Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.

Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites. Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors. In this study, we produced a single-chain variable fragment (scFv) phage display library from naive mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naive phage display antibody library to the study of F. hepatica.

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets.

Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina paired-end, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The final genome assembly is 177 Mbp with contig N50 size of 75 kbp and a scaffold N50 size of 2.3 Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts., Measurement(s)whole genome sequencing assay • transcription profiling assay • sequence_assembly • sequence annotation • mitochondrial DNATechnology Type(s)DNA sequencing • RNA sequencing • genome assembly • bioinformatics analysisSample Characteristic - OrganismHymenolepis diminuta Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10283018

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhVV) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasites global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control. Funding Agencies|Polish Ministry of Scientific Research and Information Technology [PBZ-MIN-007/PO4]

Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates. The trematode Fasciola hepatica is an important veterinary and human parasite. In this work, we infected rats with F. hepatica and amplified the generated antibody repertoire to produce a single-chain variable fragment (scFv) phage display library. The library was used to identify a scFv that recognises cathepsin L1, a major component of the adult parasites excretory/secretory material and an important vaccine candidate. This is the first report of the construction of a phage display antibody library from a F. hepatica infected host, and also the first instance such a library has been used to identify an affinity-matured monoclonal antibody fragment that binds to a F. hepatica antigen. The scFv library and methods detailed should facilitate future research characterising F. hepatica antigens.

Most animal research is less evidence-based for females, with the majority of studies conducted on males. Since immune responses vary between males and females, sexual dimorphism in immunity contributes, among other things, to sex-based differences post-vaccination. However, the issue of sex effects in animal vaccine research is rarely considered in vaccine study design. Previously, we have evaluated the efficacy of cathepsin L3 (FhCL3-1 and FhCL3-2) and B3 proteases (FhCB3) from juvenile Fasciola hepatica as vaccines against fasciolosis in male rats. Their administration resulted in reductions in liver fluke recovery in the range of 47–63% when compared with an infection control group. Here, we investigated if the protective effect of vaccination with these proteins can also be observed for female rats. The data indicates females were not protected from F. hepatica infection when vaccinated with juvenile cathepsins. Only in the FhCL3-2 vaccinated group was a low, non-significant, reduction in worm burden observed (21%). Although liver fluke mean body lengths and wet weights were reduced in vaccinated animals when compared with the infection controls, these effects were adjuvant- not vaccine-induced, while for males changes in these parameters were related primarily to vaccination. Specific humoral responses throughout the study were evident; however, trends in antibody responses in females replicated trends observed previously for male humoral responses. Formerly, elevated levels of FhCL3-1 and FhCL3-2 specific IgG1 and IgG2a were suggested to be correlated with protection. Here, despite increased and clear responses of these antibodies, protection was not observed. Hence, in the present study the roles of IgG1 and IgG2 in liver fluke reduction are questionable. Results demonstrated in our study show that observations obtained in one sex are not always applicable to the other sex. Hopefully, the findings of the study will stimulate discussion of the issue of sex impacts on post-vaccination outcomes and will encourage researchers to consider sex in their future vaccine studies.

Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (p = 0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.

Helminths and their products can suppress the host immune response to escape host defence mechanisms and establish chronic infections. Current studies indicate that macrophages play a key role in the immune response to pathogen invasion. They can be polarized into two distinct phenotypes: M1 and M2. The present paper examines the impact of the adult Hymenolepis diminuta tapeworm (HD) and its excretory-secretory products (ESP) on THP-1 macrophages. Monocytes were differentiated into macrophages and cultured with a living parasite or its ESP. Our findings indicate that HD and ESP have a considerable impact on human THP-1 macrophages. Macrophages treated with parasite ESP (with or without LPS) demonstrated reduced expression of cytokines (i.a. IL-1α, TNFα, TGFβ, IL-10) and chemokines (i.a. IL-8, MIP-1α, RANTES, and IL-1ra), while s-ICAM and CxCL10 expression rose after ESP stimulation. In addition, inflammatory factor expression rose significantly when macrophages were exposed to living parasites. Regarding induced and repressed pathways, significant differences were found between HD and ESP concerning their influence on the phosphorylation of ERK1/2, STAT2, STAT3, AMPKα1, Akt 1/2/3 S473, Hsp60 and Hck. The superior immunosuppressive properties of ESP compared to HD were demonstrated with lower levels of IL-1β, TNF-α, IL-6, IL-23 and IL-12p70 following stimulation. The presence of H. diminuta and its ESP were found to stimulate mixed M1/M2 macrophage phenotypes. Our findings indicate new molecular mechanisms involved in the response of human macrophages to tapeworm infection, this could be a valuable tool in understanding the mechanisms underlying the processes of immune regulation during cestodiasis.

DNA vaccination in large animals has often been associated with poor immunogenicity, consequently several approaches have been evaluated to enhance its efficacy. Here, we tested a cDNA encoding a phosphoglycerate kinase from Fasciola hepatica (cDNA-FhPGK/pCMV) as a vaccine against ovine fasciolosis and investigated whether a DNA prime/protein boost regime or CTLA-4 (cytotoxic lymphocyte antigen 4) mediated targeting improved DNA vaccine efficacy. No statistically significant differences in the cellular responses were seen in either vaccine trial when compared with the respective control groups. However, specific antibody responses were considerably enhanced in DNA primed/protein boosted sheep, but not among CTLA-4 targeted cDNA-FhPGK/pCMV vaccinated animals. Nevertheless, increased titers of specific IgG1 did not contribute to protection against infection, with no differences in liver fluke recoveries reported. If DNA vaccines against fasciolosis in target species are to reach the market one day, more research in this area is needed.

Helminths and their products can suppress the host immune response to escape host defense mechanisms and establish chronic infections. Current studies indicate that macrophages play a key role in the immune response to pathogen invasion. They can be polarized into two distinct phenotypes: M1 and M2. The present paper examines the impact of the adult Hymenolepis diminuta (HD) tapeworm and its excretory/secretory products (ESP) on THP-1 macrophages. Monocytes were differentiated into macrophages and cultured with a living parasite or its ESP. Our findings indicate that HD and ESP have a considerable impact on human THP-1 macrophages. Macrophages treated with parasite ESP (with or without LPS) demonstrated reduced expression of cytokines (i.e., IL-1α, TNFα, TGFβ, IL-10) and chemokines (i.e., IL-8, MIP-1α, RANTES, and IL-1ra), while s-ICAM and CxCL10 expression rose after ESP stimulation. In addition, inflammatory factor expression rose significantly when macrophages were exposed to living parasites. Regarding induced and repressed pathways, significant differences were found between HD and ESP concerning their influence on the phosphorylation of ERK1/2, STAT2, STAT3, AMPKα1, Akt 1/2/3 S473, Hsp60, and Hck. The superior immunosuppressive properties of ESP compared to HD were demonstrated with lower levels of IL-1β, TNF-α, IL-6, IL-23, and IL-12p70 following stimulation. The presence of HD and its ESP were found to stimulate mixed M1/M2 macrophage phenotypes. Our findings indicate new molecular mechanisms involved in the response of human macrophages to tapeworm infection, this could be a valuable tool in understanding the mechanisms underlying the processes of immune regulation during cestodiasis.