Echinococcus granulosus ubiquitin-conjugating enzymes (E2D2 and E2N) promote the formation of liver fibrosis in TGFß1-induced LX-2 cells.

BACKGROUND: Cystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear. METHODS: To investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored. RESULTS: Both rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFß1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration. CONCLUSIONS: Our findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis.

Effects of annexin B18 from Echinococcus granulosus sensu lato on mouse macrophages.

Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ß，IL-6，IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Enhanced protective immunity against Baylisascaris schroederi infection in mice through a multi-antigen cocktail vaccine approach.

Baylisascaris schroederi is among the most severe intestinal nematodes affecting giant pandas. Developing effective and secure vaccines can be used as a novel strategy for controlling repeated roundworm infection and addressing drug resistance. In our previous study, three recombinant antigens (rBsHP2, rBsGAL, and rBsUP) exhibited promising effects against B. schroederi infection in the mice model. This study extends the findings by formulating four-form cocktail vaccines (GAL+UP, HP2+UP, GAL+HP2, and GAL+HP2+UP) using three B. schroederi recombinant antigens to improve protection in mice further. Additionally, the protective differences after immunizing mice with different doses of cocktail antigens (150 µg, 100 µg, and 50 µg) were analyzed. Administration of rBs(GAL+UP), rBs(HP2+UP), rBs(GAL+HP2), and rBs(GAL+HP2+UP) significantly reduced liver and lung lesions, along with a decrease in L3 larvae by 83.7%, 82.1%, 76.4%, and 75.1%, respectively. These vaccines induced a Th1/Th2 mixed immunity, evidenced by elevated serum antibody levels (IgG, IgG1, IgG2a, IgE, and IgA) and splenocyte cytokines [interferon gamma (IFN-γ), interleukin (IL)-5, and IL-10]. Furthermore, varying cocktail vaccine dosages did not significantly affect protection. The results confirm that a 50 µg rBs(GAL+UP) dosage holds promise as a better candidate vaccine combination against B. schroederi infection, providing a basis for developing the B. schroederi vaccine.

Protective efficacy of six recombinant proteins as vaccine candidates against Echinococcus granulosus in dogs.

BACKGROUND: Cystic echinococcosis (CE) is caused by the infection of Echinococcus granulosus sensu lato (E. granulosus s.l.), one of the most harmful zoonotic helminths worldwide. Infected dogs are the major source of CE transmission. While praziquantel-based deworming is a main measure employed to control dog infections, its efficacy is at times compromised by the persistent high rate of dog re-infection and the copious discharge of E. granulosus eggs into the environment. Therefore, the dog vaccine is a welcome development, as it offers a substantial reduction in the biomass of E. granulosus. This study aimed to use previous insights into E. granulosus functional genes to further assess the protective efficacy of six recombinant proteins in dogs using a two-time injection vaccination strategy. METHODS: We expressed and combined recombinant E. granulosus triosephosphate isomerase (rEgTIM) with annexin B3 (rEgANXB3), adenylate kinase 1 (rEgADK1) with Echinococcus protoscolex calcium binding protein 1 (rEgEPC1), and fatty acid-binding protein (rEgFABP) with paramyosin (rEgA31). Beagle dogs received two subcutaneous vaccinations mixed with Quil-A adjuvant, and subsequently orally challenged with protoscoleces two weeks after booster vaccination. All dogs were sacrificed for counting and measuring E. granulosus tapeworms at 28 days post-infection, and the level of serum IgG was detected by ELISA. RESULTS: Dogs vaccinated with rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31 protein groups exhibited significant protectiveness, with a worm reduction rate of 71%, 57%, and 67%, respectively, compared to the control group (P < 0.05). Additionally, the vaccinated groups exhibited an inhibition of worm growth, as evidenced by a reduction in body length and width (P < 0.05). Furthermore, the level of IgG in the vaccinated dogs was significantly higher than that of the control dogs (P < 0.05). CONCLUSION: These verified candidates may be promising vaccines for the prevention of E. granulosus infection in dogs following two injections. The rEgTIM&rEgANXB3 co-administrated vaccine underscored the potential for the highest protective efficacy and superior protection stability for controlling E. granulosus infections in dogs.

Protection against Eimeria intestinalis infection in rabbits immunized with the recombinant elongation factors EF1α and EFG.

Eimeria intestinalis is the most pathogenic species of rabbit coccidiosis, causing weight loss, diarrhea, and even acute death. The currently used anticoccidial drugs against E. intestinalis in rabbits are associated with drug resistance and residues. Immunological control might be a potential alternative. We cloned and expressed the E. intestinalis recombinant EF1α and EFG (rEi-EF1α and rEi-EFG, respectively). Rabbits were immunized subcutaneously every 14 days with 100 µg of rEi-EF1α and rEi-EFG and followed by 5 × 104 E. intestinalis sporulated oocysts orally challenge. Serum samples were collected every 7 days to measure the levels of specific antibodies and cytokines. On post-challenge day 14, rabbits were sacrificed and the anticoccidial index was evaluated. The rabbits of PBS challenged groups exhibited anorexia, diarrhea, marked intestinal wall thickening, and white nodules that formed patches, while rabbits from the rEi-EF1α or rEi-EFG challenged group exhibited milder symptoms. The rEi-EF1α group showed a 75.18% oocyst reduction and 89.01%wt gain; the rEi-EFG group had a 60.58% oocyst reduction and 56.04%wt gain. After vaccination, specific IgG levels increased and stayed high (P < 0.05). The IL-4 and IL-2 levels of rEi-EF1α immunized groups showed a significant increase after immunization (P < 0.05). Both rEi-EF1α and rEi-EFG could induce humoral and cellular immune responses. In contrast, rabbits immunized with rEi-EF1α were better protected from challenge by E. intestinalis than rEi-EFG.

Molecular characterization and functional implications on mouse peripheral blood mononuclear cells of annexin proteins from Echinococcus granulosus sensu lato.

BACKGROUND: Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. METHODS: Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). RESULTS: In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. CONCLUSIONS: Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells.

Assessment of the Immunoprotective Efficacy of Recombinant 14-3-3 Protein and Dense Granule Protein 10 (GRA10) as Candidate Antigens for Rabbit Vaccines against Eimeria intestinalis.

Eimeria intestinalis infects rabbits, causing severe intestinal coccidiosis. Prolonged anticoccidial drug use might lead to coccidia resistance and drug residues in food. Thus, vaccines are required to control rabbit coccidiosis. In this study, recombinant E. intestinalis 14-3-3 and GRA10 proteins (rEi-14-3-3 and rEi-GRA10) were obtained via prokaryotic expression and used as recombinant subunit vaccines. Fifty 30-day-old rabbits were randomly grouped as follows: PBS-uninfected group, PBS-infected group, Trx-His-S control group, and rEi-14-3-3 and rEi-GRA10 immunized groups. The rabbits were subcutaneously immunized twice at 2-week intervals, challenged with 7 × 104 sporulated oocysts, and sacrificed 14 days later. The protective effects were assessed via clinical signs, relative weight gain, oocyst reduction, mean intestinal lesion score, ACI (anticoccidial index), cytokine, and specific antibody levels in sera. The rEi-14-3-3 and rEi-GRA10 groups had higher relative weight gain rates of 81.94% and 73.61% (p < 0.05), and higher oocyst reduction rates of 86.13% and 84.87% (p < 0.05), respectively. The two immunized groups had fewer intestinal lesions (p < 0.05) and higher IgG levels (p < 0.05). Higher levels of IL-2, IL-4, and IFN-γ cytokines in the rEi-14-3-3 group (p < 0.05) and a higher level of IFN-γ in the rEi-GRA10 group (p < 0.05) were observed. The ACI values of the rEi-14-3-3 and rEi-GRA10 groups were 168.24 and 159.91, with good and moderate protective effects, respectively. Both rEi-14-3-3 and rEi-GRA10 induced humoral immunity in the rabbits. In addition, rEi-14-3-3 induced Th1- and Th2-type immune responses. Both recombinant proteins were protective against E. intestinalis infection in rabbits, with rEi-14-3-3 showing a better protective effect.

Evaluation of the immunoprotective effects of eight recombinant proteins from Baylisascaris schroederi in mice model.

BACKGROUND: Baylisascaris schroederi is the most common and harmful intestinal parasitic nematode of giant pandas, causing ascariasis. Although drug deworming is the main measure to control ascariasis in captive giant pandas, prolonged and repeated use of deworming drugs might induce resistance in nematodes and drug residues in giant pandas. Therefore, developing a safe and effective vaccine might provide a novel strategy to prevent ascariasis in captive giant pandas. METHODS: Four highly expressed secretome genes encoding excretory and secretory proteins of B. schroederi, including transthyretin-like protein 46 (BsTLP), uncharacterized protein (BsUP), hypothetical protein 1 (BsHP1), and hypothetical protein 2 (BsHP2) and four functional genes [(encoding Galectin (BsGAL), glutathione S-transferase (BsGST), fatty acid-binding protein (BsFABP), and thioredoxin peroxidase (BsTPX)] were identified based on genome and transcriptome databases of B. schroederi and used to construct recombinant proteins via prokaryotic expression. Kunming mice were vaccinated subcutaneously twice with the recombinant proteins (50 µg/mouse) mixed with Quil A adjuvant with a 2-week interval and then orally challenged with 3000 infective eggs. The immunoprotective effects of the eight recombinant proteins on mice were assessed comprehensively using surface lesion histology scores of the mouse liver and lung, larval worm reduction, serum antibody levels (IgG, IgE, IgA, IgG1, and IgG2a), and cytokine production [interferon gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-5, and IL-10]. RESULTS: Mice vaccinated with recombinant (r)BsUP (76.5%), rBsGAL (74.7%), and rBsHP2 (71.5%) showed a significant (P < 0.001) reduction in the larval worm rate compared with that in the adjuvant control. Besides, the surface lesions in the liver and lung of the vaccinated mice were alleviated. Serum levels of total IgG, IgE, IgA, IgG1, IgG2a, and cytokines, including IL-10, IL-5, and IFN-γ, were significantly higher (P < 0.001) than those in the control group. CONCLUSIONS: The results showed that candidate three vaccines (rBsUP, rBsGAL, and rBsHP2) could provide effective protection against egg infection in mice associated with a mixed Th1/2-type immune response.

Protective efficacy of recombinant proteins AMA1 and IMP1 in rabbits infected with Eimeria intestinalis.

Eimeria intestinalis is one of the most pathogenic rabbit coccidia species causing severe intestinal damage and increased risk of secondary infection from opportunistic pathogens, which results in huge economic losses to the rabbit industry. Anticoccidial drugs are currently the main method to control coccidiosis; however, increasing resistance and drug residues have fueled research on anticoccidial vaccines. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1), as surface proteins, are associated with host invasion and might have the potential as candidate vaccine antigens. In the present study, recombinant IMP1 (rEiIMP1) and AMA1 (rEiAMA1) from E. intestinalis were expressed using Escherichia coli BL21. The immunoreactivity and immunoprotective effects of rEiIMP1 and rEiAMA1 were then analyzed. Fifty rabbits were grouped randomly (n = 10 per group): The unimmunized-unchallenged control group (sterilized phosphate-buffered saline (PBS)), the unimmunized-challenged control group (sterilized PBS), the vector protein-challenged control group (100 µg of pET-32a vector protein per rabbit), the rEiIMP1 immunized group (100 µg of rEiIMP1 per rabbit), and the rEiAMA1 immunized group (100 µg of rEiAMA1 per rabbit). After two immunizations, the rabbits were challenged with homologous oocysts (except for the unimmunized-unchallenged group). Serum specific antibody levels were assessed weekly throughout the experimental period; and the levels of different cytokines in the serum before the challenge were detected. The clinical symptoms, oocysts output, weight gain, feed conversion ratio (FCR), and lesion scores were recorded after experimental infection, and the anticoccidial indexes (ACIs) were calculated. The results showed that both rEiIMP1 and rEiAMA1 had good immunoreactivity. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed 66.74 % and 63.14 % oocyst reduction, respective land 81.79 % and 78.87 % body weight gain, respectively. The rEiIMP1 and rEiAMA1 groups had lower FCRs (3.77:1 and 4.06:1, respectively) and lesion scores (P = 0.00). The rEiIMP1 and rEiAMA1 showed moderate effects, with an ACI of 152.09 and 147.17, respectively. Immunization induced high levels of anti-rEiIMP1 and -rEiAMA1 antibodies. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed significantly increased interleukin (IL)- 2 (P = 0.00), interferon gamma (IFN)- γ (P = 0.00), and IL- 4 (P = 0.00) levels. Therefore, this study provided potential candidate vaccine antigens for E. intestinalis.

Transcriptome mining of RNA viruses (family Totiviridae ) in Eimeria necatrix and Eimeria stiedai.

Coccidian protozoa from the genus Eimeria are widespread parasites of vertebrates, causing serious disease (coccidiosis) and economic loss most notably in poultry. Several species of Eimeria are themselves infected with small RNA viruses assigned to the family Totiviridae . In this study, the sequences of two such viruses were newly determined, one of which represents the first complete protein-coding sequence of a virus from E. necatrix , an important pathogen of chickens, and the other of which is from E. stiedai , an important pathogen of rabbits. Sequence features of the newly identified viruses, compared with those of ones reported previously, provide several insights. Phylogenetic analyses suggest that these eimerian viruses constitute a well-demarcated clade, probably deserving of recognition as a distinct genus.

Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercus tenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Comprehensive molecular characterization of complete mitogenome assemblies of 33 Eimeria isolates infecting domestic chickens.

BACKGROUND: Coccidiosis caused by Eimeria is one of the most severe chicken diseases and poses a great economic threat to the poultry industry. Understanding the evolutionary biology of chicken Eimeria parasites underpins development of new interactions toward the improved prevention and control of this poultry disease. METHODS: We presented an evolutionary blueprint of chicken coccidia by genetically characterizing complete mitogenome assemblies of 33 isolates representing all seven known Eimeria species infecting chickens in China. Further genome- and gene-level phylogenies were also achieved to better understand the evolutionary relationships of these chicken Eimeria at the species level. RESULTS: 33 mitogenomes of chicken eimerian parasites ranged from 6148 bp to 6480 bp in size and encoded typical mitochondrial compositions of apicomplexan parasites including three protein-coding genes (PCGs), seven fragmented small subunit (SSU) and 12/13 fragmented large subunit (LSU) rRNAs. Comparative genomics provided an evolutionary scenario for the genetic diversity of PCGs-cytochrome c oxidase subunits 1 and 3 (cox1 and cox3) and cytochrome b (cytb); all were under purifying selection with cox1 and cox3 being the lowest and highest evolutionary rates, respectively. Genome-wide phylogenies classified the 33 Eimeria isolates into seven subgroups, and furthermore Eimeria tenella and Eimeria necatrix were determined to be more closely related to each other than to the other eight congenic species. Single/concatenated mitochondrial protein gene-based phylogenies supported cox1 as the genetic marker for evolutionary and phylogenetic studies for avain coccidia. CONCLUSIONS: To our knowledge, these are the first population-level mitogenomic data on the genus Eimeria, and its comprehensive molecular characterization provides valuable resources for systematic, population genetic and evolutionary biological studies of apicomplexan parasites in poultry.

Macrophage Migration Inhibitory Factor in Psoroptes ovis: Molecular Characterization and Potential Role in Eosinophil Accumulation of Skin in Rabbit and Its Implication in the Host-Parasite Interaction.

Psoroptes ovis, a common surface-living mite of domestic and wild animals worldwide, results in huge economic losses and serious welfare issues in the animal industry. P. ovis infestation rapidly causes massive eosinophil infiltration in skin lesions, and increasing research revealed that eosinophils might play an important role in the pathogenesis of P. ovis infestation. Intradermal injection of P. ovis antigen invoked massive eosinophil infiltration, suggesting that this mite should contain some relative molecules involved in eosinophil accumulation in the skin. However, these active molecules have not yet been identified. Herein, we identified macrophage migration inhibitor factor (MIF) in P. ovis (PsoMIF) using bioinformatics and molecular biology methods. Sequence analyses revealed that PsoMIF appeared with high similarity to the topology of monomer and trimer formation with host MIF (RMSD = 0.28 angstroms and 2.826 angstroms, respectively) but with differences in tautomerase and thiol-protein oxidoreductase active sites. Reverse transcription PCR analysis (qRT-PCR) results showed that PsoMIF was expressed throughout all the developmental stages of P. ovis, particularly with the highest expression in female mites. Immunolocalization revealed that MIF protein located in the ovary and oviduct of female mites and also localized throughout the stratum spinosum, stratum granulosum, and even basal layers of the epidermis in skin lesions caused by P. ovis. rPsoMIF significantly upregulated eosinophil-related gene expression both in vitro (PBMC: CCL5, CCL11; HaCaT: IL-3, IL-4, IL-5, CCL5, CCL11) and in vivo (rabbit: IL-5, CCL5, CCL11, P-selectin, ICAM-1). Moreover, rPsoMIF could induce cutaneous eosinophil accumulation in a rabbit model and increased the vascular permeability in a mouse model. Our findings indicated that PsoMIF served as one of the key molecules contributing to skin eosinophil accumulation in P. ovis infection of rabbits.

Artificial Infestation of Sarcoptes scabiei (Acari: Sarcoptidae) in Rabbits Exhibits Progressive Pathological Changes, Apoptosis, and Keratinization in the Skin.

Sarcoptes scabiei (S. scabiei) is an ectoparasite that can infest humans and 150 mammalian host species, primarily causing pruritus, crust, and alopecia. However, neither the pathological process of host skin under S. scabiei infection nor the mechanism of S. scabiei infection in regulating apoptosis and keratinization of host skin has been studied yet. In this study, a total of 56 rabbits were artificially infested with S. scabiei, and the skin samples were collected at seven different time points, including 6 h, 12 h, day 1, day 3, 1 week, 4 weeks, and 8 weeks, whereas a group of eight rabbits served as controls. We measured epidermal thickness by H&E staining, observed the skin ultrastructure by electron microscopy, and detected the degree of skin apoptosis by TUNEL staining. The level of transcription of genes related to apoptosis and keratinization was detected by quantitative real-time PCR (qRT-PCR), and the level of Bcl-2 protein expression was further detected using immunohistochemistry. Our results showed that, with increased infestation time, the epidermal layer of the rabbit skin exhibited significant thickening and keratinization, swollen mitochondria in the epidermal cells, and increased skin apoptosis. The level of caspase-1, 3, 8, 10, 14, and Bcl-2 mRNA expression was increased, whereas the level of keratin 1 and 5 was decreased after S. scabiei infestation. In conclusion, S. scabiei infestation causes thickening of the epidermis, which may be related to apoptosis-induced proliferation and skin keratinization.

Vaccination with a cocktail vaccine elicits significant protection against Sarcoptes scabiei in rabbits, whereas the multi-epitope vaccine offers limited protection.

Sarcoptes scabiei cause scabies in humans or sarcoptic mange in animals. Currently, information regarding vaccines against S. scabiei is limited and no commercial vaccine is available. In present study, we expressed and mixed recombinant S. scabiei serpin (rSs-serpin), recombinant S. scabiei chitinase-like protein-5 [rSs-CLP5] and -12 [rSs-CLP12] as a cocktail vaccine (three proteins mixed), and also a multi-epitope protein derived from these three S. scabiei genes was expressed as a vaccine candidate to evaluate the effects of two vaccine strategies. Four test groups (n = 12 per group) and a control group (n = 12 per group) were involved in this vaccination trial. The results showed that 91.67% (11/12) and 83.33% (10/12) of rabbits exhibited no detectable skin lesions from S. scabiei infestation in cocktail vaccine groups, whereas two multi-epitope groups produced only a few rabbits (5/12, 6/12) having no detectable skin lesions. Four test groups displayed significant increases in specific IgG antibodies (Abs) and total IgE Abs after immunized with recombinant proteins. Taken together, our data demonstrated a mixture of rSs-serpin, rSs-CLP5 and rSs-CLP12 was a promising vaccine candidate that induced robust immune protection and could significantly decrease mite populations to reduce the direct transmission between rabbits. However, vaccination with the multi-epitope protein showed limited protection in rabbits.

Evaluation of the immune protective effects of rEmMIC2 and rEmMIC3 from Eimeria magna in rabbits.

Eimeria magna is a common pathogen in rabbits, which results in lethargy, weight loss, diarrhea, and even death in severe cases after infection. The current method for preventing rabbit coccidiosis is to add anticoccidial drugs to the diet. However, there are many concerns about drug resistance and drug residues. In our study, the rEmMIC2 and rEmMIC3 proteins were cloned and expressed to evaluate potential as recombinant subunit vaccine candidate antigens. The protective effects of rEmMIC2 and rEmMIC3 were evaluated by the relative weight gain ratio, oocyst decrease rate, anticoccidial index, feed conversion ratio, pathological alterations, clinical symptoms, specific IgG antibody, and cytokine levels in rabbits. The molecular weights of rEmMIC2 and rEmMIC3 were 18.69 kDa and 17.47 kDa, respectively. After the coccidia challenge, the control groups showed anorexia and soft poop, whereas the experimental group showed few anorexia symptoms. Significantly different from the control group, the relative weight gain ratios of the immunized rEmMIC2 and rEmMIC3 groups were 78.37% and 75.29%, respectively, and the oocyst reduction was 77.95% and 76.09%, respectively, and the anticoccidial index was 171.12 and 169.29, respectively. IgG antibody, IFN-γ, IL-4, IL-10, and IL-17 levels were significantly increased in the experimental group. The results showed that rEmMIC2 and rEmMIC3 have potential as vaccine candidate antigens.

Immunomodulatory effects of two recombinant arginine kinases in Sarcoptes Scabiei on host peripheral blood mononuclear cells.

Background: As an important zoonotic parasitic disease with global distribution, scabies causes serious public health and economic problems. Arginine kinase (AK) is involved in cell signal transduction, inflammation, and apoptosis. Two AKs were identified in Sarcoptes scabiei, but their functions in the host immune response remain unclear. Methods: rSsAK-1 and rSsAK-2 were expressed, purified, and immunolocalized. The effects of rSsAK-1 and rSsAK-2 on rabbit PBMC proliferation, apoptosis, and migration; Bcl-2, Bcl-xl, Fas, Bax, and NF-κB transcription levels; and IL-2, IFN-γ, IL-4, IL-10, TGF-ß1, and IL-17 secretion were detected. Results: rSsAK-1 and rSsAK-2 were cloned and expressed successfully. Both enzymes were ~57 kDa and contained 17-kDa tagged proteins, and had good catalytic activity and immunoreactivity. The proteins were located in the S. scabiei exoskeleton, chewing mouthparts, legs, stomach, and intestine. SsAK-1 and SsAK-2 were secreted in the pool and epidermis of the skin lesions, which may be involved in S. scabiei-host interaction. rSsAK-1 and rSsAK-2 significantly promoted cell proliferation, induced cell migration, inhibited apoptosis, and increased Bcl-2, Bcl-xl and NF-κB (p65) transcription levels concentration-dependently, and inhibited IL-2, IFN-γ, and IL-10 secretion and promoted IL-4 and IL-17 secretion. Conclusion: rSsAK-1 and rSsAK-2 might increase Bcl-2 and Bcl-xl expression by activating the NF-κB signaling pathway to promote cell proliferation and inhibit apoptosis, which induced PBMC survival. By inducing PBMC migration to the infection site, rSsAK-1 and rSsAK-2 shifted the Th1/Th2 balance toward Th2 and changed the Th17/Treg balance, which indicated their immune role in S. scabiei allergic inflammation.

Effects of Sarcoptes scabiei Translationally Controlled Tumor Protein (TCTP) on Histamine Release and Degranulation of KU812 Cells.

Scabies is a common parasitic dermatological infection worldwide that is often neglected. Scabies mites stimulate host inflammatory symptoms via secreted and excreted proteins, which induce basophil and mast cell degranulation and host histamine release. However, the mechanism of degranulation and histamine release is unclear. Moreover, the Sarcoptes scabiei translationally controlled tumor protein (TCTP) is predicted as an excreted protein, which may be involved in host inflammatory response regulation. First, we evaluated S. scabiei TCTP gene (SsTCTP) transcription in larvae, nymphs, and adults by qRT-PCR, and SsTCTP transcription was highest in larvae, followed by nymphs. Second, we found that the S. scabiei TCTP recombinant protein (rSsTCTP) promoted mice histamine release in vivo by Evans blue Miles assay. Therefore, to further explore the possible role of S. scabiei TCTP in host inflammatory response regulation, we established a degranulation model of KU812 cells. The results of the degranulation model suggested that rSsTCTP could induce enhanced degranulation of KU812 cells and increase the secretion of histamine and the expression of IL-4, IL-6, and IL-13 in vitro. In conclusion, we speculate that scabies mites could stimulate host histamine release and Th2 response by excreting S. scabiei TCTP.

Comprehensive Molecular Characterization of the Mitochondrial Genome of the Takin Lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae).

The takin lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae) causes lethal bronchopneumonia and represents severe threats to captive and wild populations. However, until now there has been very limited information available concerning the molecular epidemiology and evolutionary biology of V. eleguneniensis. Mitochondrial genomes (mtDNAs) can provide resources for investigations in these areas and, therefore, can assist with the surveillance and control of this lungworm. Herein, the complete mtDNA of V. eleguneniensis was sequenced and characterized with Illumina pipeline analyses. This circular genome (13,625 bp) encoded twelve protein-coding genes (PCGs), two rRNAs, and twenty-two tRNAs, with notable levels of AT and GC skews. Comparative genomics revealed a purifying selection among PCGs, with cox1 and nad6 having the lowest and the highest evolutionary rate, respectively. Genome-wide phylogenies showed a close relationship between V. eleguneniensis and Protostrongylus rufescens in Strongylida. Single gene (PCGs or rRNAs)-based phylogenies indicated that cox1 and nad5 genes shared the same family-level topology with that inferred from genomic datasets, suggesting that both genes could be suitable genetic markers for evolutionary and phylogenetic studies of Strongylida species. This was the first mtDNA of any member of the genus Varestrongylus, and its comprehensive molecular characterization represents a new resource for systematic, population genetic and evolutionary biological studies of Varestrongylus lungworms in wildlife.