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.

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.

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.

The Molecular Basis of Ubiquitin-Conjugating Enzymes (E2s) as a Potential Target for Cancer Therapy.

Ubiquitin-conjugating enzymes (E2s) are one of the three enzymes required by the ubiquitin-proteasome pathway to connect activated ubiquitin to target proteins via ubiquitin ligases. E2s determine the connection type of the ubiquitin chains, and different types of ubiquitin chains regulate the stability and activity of substrate proteins. Thus, E2s participate in the regulation of a variety of biological processes. In recent years, the importance of E2s in human health and diseases has been particularly emphasized. Studies have shown that E2s are dysregulated in variety of cancers, thus it might be a potential therapeutic target. However, the molecular basis of E2s as a therapeutic target has not been described systematically. We reviewed this issue from the perspective of the special position and role of E2s in the ubiquitin-proteasome pathway, the structure of E2s and biological processes they are involved in. In addition, the inhibitors and microRNAs targeting E2s are also summarized. This article not only provides a direction for the development of effective drugs but also lays a foundation for further study on this enzyme in the future.

Comparative analysis of the allergenic characteristics and serodiagnostic potential of recombinant chitinase-like protein-5 and -12 from Sarcoptes scabiei.

BACKGROUND: Scabies is caused by burrowing of the mite Sarcoptes scabiei into the stratum corneum. Currently, diagnosis via routine skin scraping is very difficult, and information on the allergenic identification of S. scabiei remains limited. METHODS: We performed comparative analysis of the serological diagnostic potential of recombinant S. scabiei chitinase-like protein-5 (rSsCLP5) and recombinant S. scabiei chitinase-like protein-12 (rSsCLP12) by measuring the levels of serum-specific IgG and IgE antibodies (Abs) as diagnostic markers. In addition, the allergenic characteristics of rSsCLP5 and rSsCLP12 were evaluated using IgE-binding experiments and skin tests. RESULTS: The IgE Abs-based indirect enzyme-linked immunosorbent assay (ELISA) methods showed high sensitivity and specificity: the rSsCLP5-based assay had 93.5% sensitivity and 94.4% specificity; the rSsCLP12-based assay had 100% sensitivity and 98.1% specificity. The specific IgE Abs in infested mouse sera could bind rSsCLP5 and rSsCLP12. In skin tests, rabbits in the rSsCLP5 and rSsCLP12 groups and positive control (histamine) groups exhibited allergic reactions. Most test sites in the rSsCLP12 group had edema, bleeding spots, and even ulcers or scabs, but such allergy symptoms were rare in the rSsCLP5 group. Moreover, the allergic history rabbit group had more severe allergic reactions and lower levels of IgE Abs compared to the healthy rabbit group in the same protein group. CONCLUSIONS: These findings validate the use of IgE Abs to rSsCLP5 and rSsCLP12 as potentially useful markers for diagnosing scabies. Moreover, both rSsCLP5 and rSsCLP12 have allergenic properties, and the potential allergen rSsCLP12 is a stronger allergen than rSsCLP5.

Molecular characterization and serodiagnostic potential of two serpin proteins in Psoroptes ovis var. cuniculi.

BACKGROUND: Psoroptes ovis var. cuniculi is a common ectoparasite of wild and domestic rabbits worldwide that causes economically devastating losses in commercial rabbit husbandry and significantly affects the overall health of rabbits. Serine proteinase inhibitor (serpin) is present in almost all organisms that are involved in host-pathogen interactions, inflammatory responses, and reproductive development, among others. However, very little research has been carried out on P. ovis var. cuniculi serpins. METHODS: Two serpin genes of P. ovis var. cuniculi (Pso c 27 and PsoSP2 cDNAs) were cloned and molecularly characterized. The transcriptional profiles and tissue localization of these two serpins in P. ovis var. cuniculi were investigated by quantitative real-time PCR and immunohistochemistry, respectively. The potential function of recombinant Pso c 27 and PsoSP2 (rPso c 27 and rPsoSP2) in the serodiagnosis of P. ovis var. cuniculi infestation in rabbits was evaluated using a newly devleoped indirect enzyme-linked immunosorbent assay. RESULTS: Both the 523-residue Pso c 27 and the 240-residue PsoSP2 proteins contained typical serpin domains and signatures. Both Pso c 27and PsoSP2 cDNAs were expressed throughout the life-cycle; specifically, the cDNAs showed significantly higher expression in female mites than in larva, nymph, and male mites (Pso c 27: F(3, 8) = 1935.953, P < 0.0001; PsoSP2: F(3, 8) = 660.669, P < 0.0001). The native Pso c 27 and PsoSP2 proteins localized in the ovary and mouthparts of adult female mites, respectively. Compared to rPsoSP2, rPso c 27 showed better diagnostic efficiency, with higher values of sensitivity, specificity, and area under the receiver operating characteristic curve (rPso c 27 vs rPsoSP2: 96.0 vs 90.0%; 90.91 vs 78.18%; 0.988 vs 0.964, respectively). Moreover, rPso c 27 showed seropositivity in 80% of the rabbits as early as the 2 weeks post-infestation, prior to visible clinical signs and microscopy-positive of skin scrapings. CONCLUSIONS: These results suggest that these two serpins may play essential roles in reproductive development, serum-feeding, and pathogenicity of P. ovis var. cuniculi. Compared to PsoSP2, Pso c 27 appears to be a potential antigen for serodiagnosis of P. ovis var. cuniculi infestation in rabbits, especially at the early stage of infestation.

Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai.

Eimeria stiedai is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 (EsMIC1) and microneme protein 3 (EsMIC3) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post E. stiedai infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai.

Molecular and Functional Characterization of Inhibitor of Apoptosis Proteins (IAP, BIRP) in Echinococcus granulosus.

The larval stage of Echinococcus granulosus sensu lato, resulting in cystic echinococcosis, a parasitic zoonosis, causes huge economic losses to the livestock industry and poses a threat to public health. Inhibitor of apoptosis proteins (IAPs) is a class of endogenous anti-apoptotic family, which plays a significant functional role in the regulation of organism's development. Herein, to explore potential functions of IAPs in E. granulosus, two members of IAPs from E. granulosus (Eg-IAP and Eg-BIRP) were cloned, expressed, and molecularly characterized. Eg-IAP and Eg-BIRP encoded putative 331 and 168 residue proteins, respectively. Bioinformatic analysis showed that both proteins contained a type II BIR domain-the essential functional domain of IAPs. Fluorescence immunohistochemistry revealed that both proteins were ubiquitously localized in all life-cycle stages of E. granulosus. Our fluorescent quantitative PCR (RT-qPCR) results revealed relatively higher transcription levels of two Eg-IAPs in protoscoleces (PSCs) compared to the 18-day strobilated worms. We further used different concentrations of LCL161, a Smac-mimetic pan-IAPs inhibitor, to induce the apoptosis in PSCs in vitro, and revealed that the survival rate of PSCs and transcription levels of both genes were negatively correlated with the concentration of LCL161. While the results of light microscopy, transmission electron microscopy (TEM), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay also showed a higher apoptotic rate in PSCs with the increasing concentrations of LCL161. Taken together, our findings provide the reasonable evidence that both Eg-IAP and Eg-BIRP have potential implication in critical anti-apoptotic roles during the development of E. granulosus.

An Antibody Persistent and Protective Two rSsCLP-Based Subunit Cocktail Vaccine against Sarcoptes scabiei in a Rabbit Model.

Scabies is a highly contagious disease caused by Sarcoptes scabiei which burrows into stratum corneum of host's skin. In this study, after optimizing vaccination schedule, a vaccination trial is comprised of three test groups of rabbits (n = 10/group) by immunization with (1) rSsCLP5; (2) rSsCLP12; or (3) a mixture of rSsCLP5 and rSsCLP12, three biological replicates groups (n = 10/group) and three control groups (n = 10/group). Levels of specific IgG, total IgE and cytokines in sera were detected and histopathologically analyzed as indicators of vaccine effects. The results showed that 85% (17/20) of rabbits exhibited no detectable skin lesions of S. scabiei infestation in mixed protein groups compared to single protein groups with 75% (15/20) and 70% (14/20), respectively. Moreover, the deworming rates of mixed groups are increased by 10%-20% compared with that of single groups. Each of six groups immunized with rSsCLP displayed significant increases of specific IgG, total IgE, IL-10, and TNF-α. The degree of skin damage in test groups also significantly lower than that of control groups. Thus, purified rSsCLP5 and rSsCLP12 subunit cocktail vaccine induced robust immune protection and could significantly decrease mite populations to reduce the direct transmission between rabbits.

Comparative analysis of host resistance to Sarcoptes scabiei var. cuniculi in two different rabbit breeds.

BACKGROUND: Scabies, caused by infestation of the mite Sarcoptes scabiei, is one of the most severe ectoparasitic diseases in rabbits. Scabies seriously affects the commercial rabbit breeding, causing severe economic losses. Host resistance to S. scabiei is an important factor in further development of the rabbit industry. In the present study, we compared the host resistance to S. scabiei var. cuniculi of a new breed of domestic rabbit propagated by the Sichuan Animal Sciences Academy (QiXing rabbit, QX) compared with that of a traditional rabbit breed in the domestic rabbit industry (IRA rabbit, IRA). METHODS: Both QX and IRA rabbits were experimentally infested with live S. scabiei var. cuniculi mites for 48 h. Then, during the course of four-week experimental infestation period, the body weight of rabbits was recorded every two weeks for calculating body-weight variations in comparison to the non-infested control rabbits. Skin lesions in the foot area were assessed on weekly basis and serum samples were tested weekly for the estimation of changes in the total antibody levels (IgG, IgE and IgM). Moreover, DNA extracted from the blood samples was amplified for analysis of the genetic diversity in the major histocompatibility complex, class II, DQ Alpha (MHC-DQA) gene. RESULTS: Compared to the IRA rabbits, the QX rabbits showed a significantly higher (P < 0.05) relative body weight gain compared to the non-infested control rabbits and significantly lower (P < 0.05) scores for foot skin lesions and higher levels of IgG, IgE and IgM at weeks 1 to 4, week 2 and week 1 post-infestation, respectively. Furthermore, a polymorphism site at position 103 bp of exon two of MHC-DQA gene and a different gene frequency were found between two rabbit breeds, suggesting the genetic basis for the differential host resistance to the S. scabiei var. cuniculi between two rabbit breeds. CONCLUSIONS: The QX rabbits showed higher host resistance to S. scabiei var. cuniculi compared to the IRA rabbits at the clinical, immunological and genetic levels. These results provide a reference for the breeding of rabbits with adequately improved and sustained host resistance to scabies in the domestic rabbit industry.

Cloning, expression, characterization, and immunological properties of citrate synthase from Echinococcus granulosus.

The larval stages of the tapeworm Echinococcus granulosus (Cestoda: Taeniidae) are the causative agent of cystic echinococcosis, one of the most important parasitic zoonoses worldwide. E. granulosus has a complete pathway for the tricarboxylic acid cycle (TCA), in which citrate synthase (CS) is the key enzyme. Here, we cloned and expressed CS from E. granulosus (Eg-CS) and report its molecular characterization. The localization of this protein during different developmental stages and mRNA expression patterns during H2O2 treatment were determined. We found that Eg-CS is a highly conserved protein, consisting of 466 amino acids. In western blotting assays, recombinant Eg-CS (rEg-CS) reacted with E. granulosus-positive sheep sera and anti-rEg-CS rabbit sera, indicating that Eg-CS has good antigenicity and immunoreactivity. Localization studies, performed using immunohistochemistry, showed that Eg-CS is ubiquitously expressed in the larva, germinal layer, and adult worm sections of E. granulosus. Eg-CS mRNA expression levels increased following H2O2 exposure. In conclusion, citrate synthase might be involved in the metabolic process in E. granulosus. An assessment of the serodiagnostic potential of rEg-CS based on indirect ELISA showed that, although sensitivity (93.55%) and specificity (80.49%) are high, cross-reactivity with other parasites precludes its use as a diagnostic antigen.

A chitinase-like protein from Sarcoptes scabiei as a candidate anti-mite vaccine that contributes to immune protection in rabbits.

BACKGROUND: Scabies is caused by Sarcoptes scabiei burrowing into the stratum corneum of the host's skin and is detrimental to the health of humans and animals. Vaccines are an attractive alternative to replace the acaricides currently used in their control. METHODS: In the present study, the S. scabiei chitinase-like protein 5 (SsCLP5) was characterized and recombinant SsCLP5 (rSsCLP5) was evaluated as a candidate vaccine protein for anti-mite protection in rabbits. The expression, characterization and immunolocalization of SsCLP5 were examined. Vaccination experiments were performed on three test groups (n = 12 per group) immunized with purified rSsCLP5. Control groups (n = 12 per group) were immunized with PBS, QuilA saponin or empty vector protein. After challenge, the inflammatory reaction and skin lesions were graded and rSsCLP5 indirect ELISA was used to detect antibody IgG levels in serum samples at the time of vaccination and post-challenge. RESULTS: The results showed that rSsCLP5 had high immunoreactivity and immunogenicity. In S. scabiei, SsCLP5 had a wide distribution in the chewing mouthpart, legs and exoskeleton, especially the outer layer of the exoskeleton. Vaccination with rSsCLP5 resulted in 74.3% (26/35) of rabbits showing no detectable lesions after challenge with S. scabiei. CONCLUSIONS: Our data demonstrate that rSsCLP5 is a promising candidate for a recombinant protein-based vaccine against S. scabiei. This study also provides a method for studying scabies vaccine using rabbit as an animal model and a basis for screening more effective candidate proteins.

Molecular Characterisation and Functions of Fis1 and PDCD6 Genes from Echinococcus granulosus.

Cystic echinococcosis, a parasitic zoonosis that causes significant economic losses and poses a threat to public health, is caused by larvae of the tapeworm Echinococcus granulosus. Infection causes infertile cysts in intermediate hosts that cannot produce protoscoleces (PSCs) or complete the life cycle. Herein, we cloned, expressed, and characterised mitochondrial fission protein 1 (Eg-Fis1) and programmed cell death protein 6 (Eg-PDCD6) from E. granulosus, and explored their functions related to infertile cysts. Eg-Fis1 and Eg-PDCD6 encode putative 157 and 174 residue proteins, respectively, and Western blotting indicated good reactogenicity for both. Eg-Fis1 and Eg-PDCD6 were ubiquitously distributed in all stages of E. granulosus. Furthermore, mRNAs of Eg-Fis1 and Eg-PDCD6 were upregulated following H2O2 treatment which induced apoptosis in PSCs. To investigate the regulation of apoptosis in response to oxidative stress, RNA interference (RNAi) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays were performed. The apoptotic rate of the Eg-Fis1 RNAi group was significantly lower than non-interference group, but there was no such difference for Eg-PDCD6. In conclusion, Eg-Fis1 promotes apoptosis induced by oxidative stress, whereas Eg-PDCD6 does not appear to be a key regulator of apoptosis.

Serodiagnostic Potential of Alpha-Enolase From Sarcoptes scabiei and Its Possible Role in Host-Mite Interactions.

Infestation of the epidermis with the highly contagious ectoparasite, Sarcoptes scabiei, causes scabies, which is characterized by intense itching, pruritus, and secondary infection. This condition affects humans, livestock, and wildlife worldwide, incurring large economic losses and reducing the quality of human life. In the present study, we cloned the alpha-enolase, a key enzyme in the glycolytic and gluconeogenesis pathways, from S. scabiei var. cuniculi, characterized it and produced soluble recombinant enolase protein (rSsc-eno). We determined the localization of Ssc-eno in isolated mites and mites in lesioned skin. The results showed that native enolase was intensely localized in the tegument of the mouthparts, the entire legs, and the whole mites' body, as well as in the gut and reproduction system. Interestingly, we found that native enolase was widely distributed in mites in lesioned skin, with obvious high protein intensity compared with isolated mites. Building on good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) based on rSsc-eno showed 92% sensitivity and 95.8% specificity, compared with other indirect ELISA in this study, rSsc-eno based ELISA is better in detecting scabies in rabbits. Besides, this method can detect S. scabiei infection as early as 1 week post infection. Compared with other detection methods, such as traditional microscopic examination and recently published universal conventional PCR, rSsc-eno ELISA was more effective to detect early infection in rabbits. Additionally, in vitro incubation experiments demonstrated the concentration-dependent acaricidal activity of rabbit anti-rSsc-eno sera against larval mites, suggested its potential as a vaccine candidate.

Molecular characterization and allergenicity potential of triosephosphate isomerase from Sarcoptes scabiei.

Scabies is an allergic skin disease that affects millions of mammals worldwide, including humans. It is a neglected tropical disease that represents a significant public health threat, particularly in economically disadvantaged populations. An effective vaccine is not currently available, and the exact mode of pathogenesis remains unclear. Herein, we identified, cloned and recombinantly expressed triosephosphate isomerase from Sarcoptes scabiei (S. scabiei). Immunohistochemical analyses showed that S. scabiei triosephosphate isomerase (Ss-TIM) is localized in the legs and chewing mouthparts of mites, and in infected rabbit skin (keratinized skin and embedded mites). Intradermal skin tests of rabbits injected with recombinant S. scabiei triosephosphate isomerase (rSs-TIM) revealed a flare, erythema and wheal reaction. These findings suggest that Ss-TIM may contribute to host invasion and induce an allergic response in the host.

Identification of a novel PYP-1 gene in Sarcoptes scabiei and its potential as a serodiagnostic candidate by indirect-ELISA.

Scabies is a parasitic disease caused by the ectoparasite Sarcoptes scabiei, affecting different mammalian species, including rabbits, worldwide. In the present study, we cloned and expressed a novel inorganic pyrophosphatase, Ssc-PYP-1, from S. scabiei var. cuniculi. Immunofluorescence staining showed that native Ssc-PYP-1 was localized in the tegument around the mouthparts and the entire legs, as well as in the cuticle of the mites. Interestingly, obvious staining was also observed on the fecal pellets of mites and in the integument of the mites. Based on its good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant Ssc-PYP-1 (rSsc-PYP-1) as the capture antigen was developed to diagnose sarcoptic mange in naturally infected rabbits; the assay had a sensitivity of 92·0% and specificity of 93·6%. Finally, using the rSsc-PYP-1-ELISA, the Ssc-PYP-1 antibody from 10 experimentally infected rabbits could be detected from 1 week post-infection. This is the first report of S. scabiei inorganic pyrophosphatase and the protein could serve as a potential serodiagnostic candidate for sarcoptic mange in rabbits.

Molecular characteristics and serodiagnostic potential of chitinase-like protein from Sarcoptes scabiei.

Scabies, caused by the mite Sarcoptes scabiei, is an allergic skin disease that affects millions of people and other mammals worldwide. This highly contagious parasitic disease is among the top 50 epidemic disease and is regarded as a neglected tropical disease. Diagnosis of scabies is difficult in the early stage, and the pathogenesis of scabies is not currently clear. Here, we expressed, identified and located the chitinase-like protein of S. scabiei (SsCLP), and evaluated its potential as an early-stage diagnostic antigen for rabbit scabies. Indirect ELISA using recombinant SsCLP (rSsCLP) exhibited diagnostic sensitivity of 94.4% (17/18) and specificity of 86.7% (26/30). Early diagnostic test after artificial infection of rabbits with S. scabiei for 1 week showed a positive detection rate of 96.7% (29/30). Immunolocalization assays showed that fluorescence signals were localized on the surface of mites and, in infected rabbits, were observed in keratinized skin and embedded mites. Intradermal skin tests of rabbits by injecting rSsCLP showed a wheal, flare and erythema reaction. These results suggest that S. scabiei chitinase-like protein is conducive to host invasion, participates in inducing the allergic response of the host, and is an effective antigen for the diagnosis of S. scabiei.

Expression and characterisation of a Sarcoptes scabiei protein tyrosine kinase as a potential antigen for scabies diagnosis.

Scabies is a disease that harms humans and other animals that is caused by the itch mite Sarcoptes scabiei burrowing into the stratum corneum of the skin. In the early stages of scabies, symptoms are often subclinical and there are no effective diagnostic methods. Herein, we cloned, expressed and characterised an S. scabiei protein tyrosine kinase (SsPTK) and evaluated its diagnostic value as a recombinant antigen in rabbit during the early stages of Sarcoptes infestation. The SsPTK protein is ~30 kDa, lacks a signal peptide, and shares high homology with a PTK from the rabbit ear mite Psoroptes ovis cuniculi. The protein was widely distributed at the front end of mites, particularly in the chewing mouthparts and legs. Indirect ELISA using recombinant SsPTK showed good diagnostic value, with 95.2% (40/42) sensitivity and 94.1% (48/51) specificity for detecting anti-PTK antibody in serum samples from naturally-infested rabbits. More importantly, PTK ELISA could diagnose infection in the early stages (infestation for 1 week) with an accuracy of 100% (24/24). SsPTK therefore shows potential as a sensitive antigen for the early diagnosis of parasitic mite infestation.

Molecular characterization of calmodulin from Sarcoptes scabiei.

Scabies, caused by the mite Sarcoptes scabiei, is a highly contagious parasitic disease that affects millions of people and other mammals worldwide. Calmodulin (CaM) is an important calcium sensor that participates in various critical physiological processes. In this study, the CaM of Sarcoptes scabiei (SsCaM) was cloned and expressed, and sequence analyses were performed using bioinformatics tools. Recombinant SsCaM (rSsCaM) was used to detect antigenicity using immunoblotting assays, and the serodiagnostic potential of rSsCaM was assessed by indirect enzyme-linked immuno-sorbent assay (ELISA). The calcium binding properties and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence of rSsCaM were also measured. The results indicated that SsCaM contains a 450-bp open reading frame that encodes for a polypeptide with 149 amino acids, and SsCaM was expressed as a soluble protein. Multiple sequence alignment and phylogenetic analyses indicated similarity and genetic distance between SsCaM and other species. The calcium binding properties and ANS fluorescence of rSsCaM indicated typical calcium binding characteristics. Immunolocalizaton assay showed that SsCaM was widespread in S. scabiei. SsCaM-based ELISA exhibited a sensitivity of 87.5% (28/32) and a specificity of 22.5% (9/40) for detecting anti-CaM antibodies in the sera of naturally infected rabbits. The findings of this study provide a comprehensive molecular characterization of SsCaM and suggest that rSsCaM is inappropriate for detecting S. scabiei. The results may also contribute to future studies on the molecular characteristics of the CaM of parasites.