Weimin Lai

Characterization of Sarcoptes scabiei cofilin gene and assessment of recombinant cofilin protein as an antigen in indirect-ELISA for diagnosis

BackgroundScabies impairs the health of humans and animals and causes heavy economic losses. Traditional diagnostic methods for scabies are inefficient and ineffective, and so far there is no commercial immunodiagnostic or molecular based test for scabies.MethodsHere, we used recombinant Sarcoptes scabiei cofilin protein as an antigen to establish indirect ELISA. S. scabiei cofilin is highly homologous to Dermatophagoides farinae Der f 31 allergen (90xa0% identity). The S. scabiei cofilin gene was cloned and expressed in Escherichia coli to obtain recombinant protein. Western blotting and fluorescence immunohistochemistry were carried out, and we established an indirect ELISA method and detected 33 serum samples from scabies infected rabbits and 30 serum samples from naïve rabbits.ResultsWestern blotting demonstrated that S. scabiei cofilin possessed good immunogenicity and fluorescence immunohistochemistry showed the S. scabiei cofilin is widespread in the splanchnic area of mites. In ELISA, a cut-off value of 0.188 was determined to judge experimental positive and negative serum values. Specificity and sensitivity of the ELISA were 87.9 and 83.33xa0%, respectively.ConclusionsRecombinant S. scabiei cofilin showed potential value as a diagnostic antigen. The ELISA method established could be used in clinical diagnosis and provide experimental information in minimal or asymptomatic infection.

Expression, tissue localization and serodiagnostic potential of Taenia multiceps acidic ribosomal protein P2

BackgroundThe larval stage of Taenia multiceps, also known as coenurus, is the causative agent of coenurosis, which results in severe health problems in sheep, goats, cattle and other animals that negatively impact on animal husbandry. There is no reliable method to identify coenurus infected goats in the early period of infection.MethodsWe identified a full-length cDNA that encodes acidic ribosomal protein P2 from the transcriptome of T. multiceps (TmP2). Following cloning, sequencing and structural analyses were performed using bioinformatics tools. Recombinant TmP2 (rTmP2) was prokaryotically expressed and then used to test immunoreactivity and immunogenicity in immunoblotting assays. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of rTmP2 for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, 20 goats were randomly divided into a drug treatment group and a control group. Each goat was orally given mature, viable T. multiceps eggs. The drug treatment group was given 10xa0% praziquantel by intramuscular injection 45xa0days post-infection (p.i), and all goats were screened for anti-TmP2 antibodies with the indirect ELISA method established here, once a week for 17xa0weeks p.i.ResultsThe open reading frame (366xa0bp) of the target gene encodes a 12.62xa0kDa protein, which showed high homology to that from Taenia solium (93xa0% identity) and lacked a signal peptide. Immunofluorescence staining showed that TmP2 was highly localized to the parenchymatous zone of both the adult parasite and the coenurus; besides, it was widely distributed in cystic wall of coenurus. Building on good immunogenic properties, rTmP2-based ELISA exhibited a sensitivity of 95.0xa0% (19/20) and a specificity of 96.3xa0% (26/27) in detecting anti-P2 antibodies in the sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmP2 antibody was detectable in the control group from 3 to 10xa0weeks and 15 to 17xa0weeks p.i. In the drug-treated group, the anti-TmP2 antibody dropped below the cut-off value about 2xa0weeks after treatment with praziquantel and remained below this critical value until the end of the experiment.ConclusionThe indirect ELISA method developed in this study has the potential for detection of T. multiceps infections in hosts.

Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha.

Heterakis gallinae and Heterakis beramporia are the most prevalent nematode infecting native chicken breed, causing major economic losses. In the present study, the complete mitochondrial genomes (mt) of H. gallinae and H. beramporia were amplified by long-PCR and then sequenced. The complete mt genomes of H. gallinae and H. beramporia were 13,973bp and 14,012bp in size, respectively. Both mt genomes contain 12 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. All genes are transcribed in the same direction and the gene arrangement is identical to Ascaridia spp. Phylogenetic analysis based on the 12 protein-coding genes revealed that the family Heterakidae (represented by H. gallinae and H. beramporia) was more closely related to the infraorder Ascaridomorpha than it was to the infraorder Oxyuridomorpha. The present study determined the complete mt genome sequences for two Heterakis species, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these Heterakis parasites.

GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect ELISA

BackgroundCoenurosis is caused by coenurus, the metacestode of Taenia multiceps, which mainly parasitizes the brain and spinal cord of cattle, sheep and goats. To date, no widely-approved methods are available to identify early coenurus infection.MethodsIn this study, we identified a full-length cDNA that encodes GP50 (TmGP50) from the transcriptome of T. multiceps, and then cloned and expressed in E. coli. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of recombinant TmGP50 protein (rTmGP50) for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, we orally infected 20 goats with mature T. multiceps eggs. Praziquantel (10%) was given to 10 of the goats 45xa0days post-infection (p.i.). Blood samples were collected for 17xa0weeks p.i. from the 20 goats and anti-rTmGP50 antibodies were evaluated using the indirect ELISA established here.ResultsThe TmGP50 contains an 897xa0bp open reading frame, in which signal sequence resides in 1u2009~u200948 sites and mature polypeptide consists of 282 amino acid residues. Immunofluorescence staining showed that native TmGP50 was localized to the microthrix and parenchymatous zone of the adult parasite and coenurus, and the coenurus cystic wall. The indirect ELISA based on rTmGP50 exhibited a sensitivity of 95.0% and a specificity of 92.6% when detecting GP50 antibodies in sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable from 2 to 17xa0weeks p.i. in the control group, while the antibody fell below the cut-off value about 3xa0weeks after praziquantel treatment.ConclusionOur results indicate that recombinant TmGP50 is a suitable early diagnostic antigen for coenurus infection in goats.

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.

An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats.

Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 μg/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis.

Zoonotic Baylisascaris procyonis roundworms in raccoons, China.

To the Editor: Baylisascaris procyonis, an intestinal roundworm that infects raccoons (Procyon lotor), causes fatal or severe neural larva migrans in animals and humans (1,2). Globally, ≈130 species of wild and domesticated animals are susceptible (2). Infections in humans typically occur in children who have the disorders pica or geophagia and ingest B. procyonis eggs in items contaminated with raccoon feces (3). Clinical manifestations include ocular disease, eosinophilic encephalitis, and eosinophilic cardiac pseudotumors; severe infection can lead to death. Since 1984, ≈24 cases of B. procyonis–related human neural larva migrans have been reported, mainly in the United States (1,3–5; K.R. Kazacos, pers. comm.). Despite few cases among humans, lack of effective treatment and widespread distribution of infected raccoons in close association with humans make B. procyonis a potentially serious public health threat (2,6). The current distribution of B. procyonis is poorly recorded in Asia (2,7), except for Japan (8). We describe B. procyonis infections among raccoons in China as part of a series of ongoing surveys of helminthic zoonoses linked to captive exotic animals in zoologic gardens (ZGs) in China. n nMore than 90% of raccoons in China (n >320) are raised as exotic ornamental animals in 18 ZGs. During 2011–2013, we collected 2×308 fecal samples (i.e., 1 repeat within each sampling) from 277 raccoons in 12 randomly selected ZGs (Technical Appendix Figure 1). Samples were stored in individual plastic bags at –20°C until use. We examined raccoons (n = 31) at the Sichuan ZGs twice, in June 2012 and May 2013. We identified B. procyonis eggs in feces using morphologic and molecular analyses (1,2,9). The nuclear first internal transcribed spacer (428 bp) and mitochondrial cytochrome c oxidase subunit 1 (cox-1, 938 bp) genes in each sample were PCR-amplified and sequenced. B. procyonis infection was confirmed by sequencing and phylogenetic analyses of both genes (7,9). We reexamined ≈60% of fecal samples to validate results. Prevalence (95% CI) was calculated for the overall population and independently for female, male, juvenile, and adult raccoons. We determined differences between the tested ZG prevalence and prevalence by sex or age of raccoons using χ2 or Fisher exact tests in SAS (SAS Institute, Cary, NC, USA); p values <0.05 were considered significant. n nBuilding on egg-based morphologic characterization and internal transcribed spacer 1 and cox-1 gene-based phylogenies using neighbor-joining trees (Technical Appendix Figure 2), we found B. procyonis in raccoon feces from 5/12 ZGs (42%; 95% CI 14%–70%), including 2 in the most densely populated provinces, Henan and Sichuan. More infections were found in western than central and eastern ZGs (4/6 and 1/6, respectively; Table, Technical Appendix Figure 1) (p = 0.079). Fecal samples of 35 raccoons (13%; 95% CI 9%–17%) tested positive for B. procyonis. The mean intensity of egg shedding was 5,000 eggs per gram (range 800–11,200 eggs per gram; data not shown). No significant difference was observed in the intensity of shedding by comparing sex and age of animals, and no significant differences were noted in the mean prevalence between female and male raccoons (12% versus 14%; p = 0.677) or between adult and juvenile animals (13% versus 10%; p = 0.536). n n n nTable n nPrevalence of Baylisascaris procyonis roundworm infections among captive raccoons, China, 2011–2013* n n n nThis investigation documents the presence and prevalence of B. procyonis among raccoons in China. The findings imply that raccoons harboring this parasite have the potential for spreading it to humans. One reason is that captive raccoons adapt readily to humans and easily take food offered by hand; another is that communal raccoon latrine sites in ZGs are usually close to areas where humans gather, so ZG visitors may be exposed to large numbers of eggs (Technical Appendix Figure 3). These eggs can remain viable and infective for years (2), and latrines are recognized as primary sources of transmission of B. procyonis to humans (4). Current public health initiatives to prevent B. procyonis infections in humans rely on the education of veterinary and human health care professionals, who in turn inform the public (1,6,10). Thus, veterinarians, clinicians, and public health officials in China should be more informed about this pathogen, especially in regions with large raccoon populations. n nBecause of a lack of clinical awareness of this illness and subsequent lack of early diagnosis and effective treatment, prevention of B. procyonis infection by education is essential. In addition, a strategy for eradication is needed. Heat, in the form of boiling water, steam-cleaning, or fire, is the optimal tool for killing B. procyonis eggs (2) and therefore can be used to decontaminate areas surrounding latrines. Within heavily contaminated areas, removing and then sterilizing the top few inches of surface soil with heat would be effective and practical (1,2). Among captive raccoon populations, particularly in China, regular deworming is also likely to be helpful in reducing novel and existing sources of infection (1–3). n nFinally, although no cases of human infection have been reported in China to our knowledge, physicians should consider including B. procyonis infections in their differential diagnoses of patients with indicative features: clinical (eosinophilic encephalitis, ocular disease), epidemiologic (raccoon exposure), radiologic (white matter disease), and laboratory results (blood and CNS eosinophilia) (1,10). This study lays the foundation for future steps to educate the population of China about B. procyonis infection and to create programs to prevent the spread of this disease to humans. n nTechnical Appendix: nDistribution of raccoons in China, morphological and molecular characterization of Baylisascaris procyonis parasitic roundworm eggs in captive raccoons in China, and the potential risk of human B. procyonis infection in China. n nClick here to view.(776K, pdf)

Transcriptome-microRNA analysis of Sarcoptes scabiei and host immune response

Scabies is a parasitic disease, caused by the mite Sarcoptes scabiei, and is considered one of the top 50 epidemic diseases and one the most common human skin disease, worldwide. Allergic dermatitis, including an intense itch, is a common symptom, however diagnosis is difficult and there is currently no effective vaccine. The goal of this study was to examine the immune interaction mechanism of both S. scabiei and infected hosts. mRNA-seq and microRNA-seq were conducted on the S. scabiei mite and on infected and uninfected hosts. We focused on differential expression of unigenes and microRNAs, as well as the real targets of unigenes in enriched immune signaling pathways. S. scabiei enhanced host immune function and decreased metabolism after infection, while the immune response of the host inhibited S. scabiei proliferation and metabolism signaling pathways. Differentially expressed unigenes of S. scabiei were enriched in the JAK-STAT signaling pathway and the Toll-like receptor signaling pathway. The differential expression analysis indicated that microRNAs of S. scabiei and hosts have major roles in regulating immune interactions between parasites and hosts.

Molecular identification and characterization of prohibitin from Echinococcus granulosus.

Prohibitin (PHB) is a widely distributed protein that functions as a molecular chaperone, is involved in the regulation of cell cycle, and maintains mitochondrial structure and functions of the anti-apoptosis, senescence, and proliferation. The aim of this study was to characterize PHB in Echinococcus granulosus (EgPHB), a harmful cestode parasite of humans, many livestock species, and wild animals. We found that EgPHB is a conserved SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain-containing protein, consisting of 289 amino acids, which shares 42.66–99.31xa0% identity with PHBs from other parasites and mammals. EgPHB was located mainly in the tegument issue of protoscoleces, in the inner body of adult worms, and was expressed widely in the germinal layer. This is the first report on prohibitin from E. granulosus, and EgPHB is considered to be a valuable protein to study more in the future.

Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis

The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using test serum samples. Immunolocalisation showed that T. pisiformis FABP (TpFABP) localised to the parenchyma of the bladder wall of the cysticercus and perinuclear cytoplasm of parenchyma of the adult parasite. After cloning and expression, recombinant TpFABP (rTpFABP) protein was used for serodiagnosis of T. pisiformis infection in rabbits by dot-ELISA. The antibody was detected 14 days post-infection in rabbits experimentally infected with T. pisiformis. Based on the necropsy results, the sensitivity and specificity of 169 serum samples tested by rTpFABP dot-ELISA were found to be 98.2% (54/55) and 92.1% (105/114), respectively. These data suggest that the dot-ELISA developed in this study has potential for detection of T. pisiformis infection in rabbits.