Morphological and Molecular Identification of Hard Ticks in Hainan Island, China.

Ticks are small, blood-sucking arachnids, known vectors of various diseases, and found throughout the world. They are distributed basically in almost all regions of China. At present, there is not much information regarding tick species on Hainan Island. They were subjected to morphological identification and imaging on an individual basis. Molecular phylogenetic analyses, based on cox1 and 16S rRNA genes, were utilized to identify the species and determine their approximate phylogenetic origin and genetic diversity. The genomic DNA of tick species was extracted, and cytochrome oxidase subunit 1 (cox1) and 16S ribosomal RNA (rRNA) genes were amplified and sequenced. The identification of five tick species, namely Rhipicephalus microplus, Rhipicephalus sanguineus, Rhipicephalus haemaphysaloides, Haemaphysalis cornigera and Haemaphysalis mageshimaensis, was carried out by morphological analysis. When employing the cox1 and 16S rRNA phylogenetic tree, all isolates of R. microplus from Hainan Island were classified as clade A and B, respectively. R. sanguineus was recognized as a member of the tropical lineage by phylogenetic analysis on the cox1 and 16S rRNA genes. Three phylogenetic groups of R. haemaphysaloides were recognized and found to be related closely to strains from China. H. cornigera and H. mageshimaensis formed one phylogenetic group, presumably from tick strains prevalent in Japan and China. The haplotype network analysis indicated that R. microplus is classed into 26 and 6 haplotypes, which correspond to cox1 and 16S rRNA gene assemblages, respectively. In addition, four cox1 haplotypes were detected in R. sanguineus. This is the first evidence that suggests genetic diversity, host range and geographical distribution of hard ticks in Hainan Island, China.

Clinical and epidemiological investigation of a child with asymptomatic COVID-19 infection following reoccurrence.

Objective: To investigate the case of a child infected with coronavirus disease 2019 (COVID-19) who had subsequent viral reactivation. Methods: We retrospectively analyzed the clinical manifestations, epidemiological data, laboratory and imaging examinations, treatment, and follow-up of the child. And then, we searched related literature using PubMed. Results: The 9-year-old boy was exposed to COVID-19 in Malawi and tested positive for NAT in Haikou, China. He was asymptomatic and admitted to our hospital. After six negative NATs, he was discharged from the hospital and quarantined in a hotel. His infection was reactivated again after 22 days (interval between first and last positive NATs). The cycle threshold (Ct) values of positive tests were 25 and 31, and the gene sequencing viral loads were very low. The viral strain Kenya/P2601/2020, a variant of the hCoV-19/Wuhan/IVDC-HB-01/2019 genome (GISAID accession IL: EPI_ISL_402119), was found when polymerase chain reaction enrichment was used to sequence the virus. However, people around him tested negative for COVID-19. Conclusion: First, we confirmed the reactivation of COVID-19 in a child. The risk of recurrent infection with SARS-CoV-2 was low, and the policy of strictly isolating patients carrying long-term viral ribonucleic acid should be reconsidered. The interval positivity was most likely due to incorrect sampling and/or testing methods. SGS and aB testing are recommended for children with viral reactivation. Second, SARS-CoV-2 viral reactivation cannot be ruled out. The possible mechanisms, such as prolonged infection and viral latent reactivation, need further investigation.

Human coronaviruses and therapeutic drug discovery.

BACKGROUND: Coronaviruses (CoVs) are distributed worldwide and have various susceptible hosts; CoVs infecting humans are called human coronaviruses (HCoVs). Although HCoV-specific drugs are still lacking, many potent targets for drug discovery are being explored, and many vigorously designed clinical trials are being carried out in an orderly manner. The aim of this review was to gain a comprehensive understanding of the current status of drug development against HCoVs, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MAIN TEXT: A scoping review was conducted by electronically searching research studies, reviews, and clinical trials in PubMed and the CNKI. Studies on HCoVs and therapeutic drug discovery published between January 2000 and October 2020 and in English or Chinese were included, and the information was summarized. Of the 3248 studies identified, 159 publication were finally included. Advances in drug development against HCoV, especially SARS-CoV-2, are summarized under three categories: antiviral drugs aimed at inhibiting the HCoV proliferation process, drugs acting on the host's immune system, and drugs derived from plants with potent activity. Furthermore, clinical trials of drugs targeting SARS-CoV-2 are summarized. CONCLUSIONS: During the spread of COVID-19 outbreak, great efforts have been made in therapeutic drug discovery against the virus, although the pharmacological effects and adverse reactions of some drugs under study are still unclear. However, well-designed high-quality studies are needed to further study the effectiveness and safety of these potential drugs so as to provide valid recommendations for better control of the COVID-19 pandemic.

Taxonomic integrative and phylogenetic identification of the first recorded Triatoma rubrofasciata in Zhangzhou, Fujian Province and Maoming, Guangdong Province, China.

BACKGROUND: Most species of Triatominae live exclusively in Latin America. However, one species, Triatoma rubrofasciata, has been recorded in the Americas as well as in various port areas in Africa and Asia. An increasing number of T. rubrofasciata have been reported in southern China in recent years. However, the origin of this invasive insect vector in China remains unknown, therefore, accurate identification and phylogenetic analysis of the bugs are urgently needed. METHODS: A total of seven triatomine insect specimens were found and collected from Maoming City, Guangdong Province, China (GDMM) and Zhangzhou City, Fujian Province, China (FJZZ), respectively. The obtained insect vector specimens were observed under a dissecting microscope for morphological classification and then the genomic DNA was extracted, and the 16S ribosomal RNA (rRNA), 28S rRNA as well as cytochrome oxidase subunit I (COI) genes of the species were amplified and sequenced. Subsequently, molecular phylogenetic analyses based on multiple alignments of the above genes were conducted in order to identify the species and determine the phylogenetic origin approximation accurately. RESULTS: The triatomine insects collected from GDMM and FJZZ were identified as Triatoma rubrofasciata using morphological and genetic analyses. All of the Chinese T. rubrofasciata captured in FJZZ, GDMM and other localities in southern China, together with a Vietnamese and Brazilian strain, formed a new, cohesive clade. T. rubrofasciata in GDMM and FJZZ are likely derived from strains found in Vietnam or Brazil. CONCLUSIONS: To the best of our knowledge, this is the first record of the invasive insect T. rubrofasciata, which is likely derived from strains native to Vietnam or Brazil, in both Maoming City, Guangdong Province and Zhangzhou City, Fujian Province of China. A comparison of the DNA sequences of the 16 s rRNA, 28 s rRNA and COI genes confirmed the specific identification of T. rubrofasciata, and its potential origin in China is based on the phylogenetic analyses undertaken in this study. More targeted interventions and improved entomological surveillance are urgently needed to control the spread of this haematophagous insect in China.

Infection against infection: parasite antagonism against parasites, viruses and bacteria.

BACKGROUND: Infectious diseases encompass a large spectrum of diseases that threaten human health, and coinfection is of particular importance because pathogen species can interact within the host. Currently, the antagonistic relationship between different pathogens during concurrent coinfections is defined as one in which one pathogen either manages to inhibit the invasion, development and reproduction of the other pathogen or biologically modulates the vector density. In this review, we provide an overview of the phenomenon and mechanisms of antagonism of coinfecting pathogens involving parasites. MAIN BODY: This review summarizes the antagonistic interaction between parasites and parasites, parasites and viruses, and parasites and bacteria. At present, relatively clear mechanisms explaining polyparasitism include apparent competition, exploitation competition, interference competition, biological control of intermediate hosts or vectors and suppressive effect on transmission. In particular, immunomodulation, including the suppression of dendritic cell (DC) responses, activation of basophils and mononuclear macrophages and adjuvant effects of the complement system, is described in detail. CONCLUSIONS: In this review, we summarize antagonistic concurrent infections involving parasites and provide a functional framework for in-depth studies of the underlying mechanisms of coinfection with different microorganisms, which will hasten the development of promising antimicrobial alternatives, such as novel antibacterial vaccines or biological methods of controlling infectious diseases, thus relieving the overwhelming burden of ever-increasing antimicrobial resistance.

Detection of helminths by loop-mediated isothermal amplification assay: a review of updated technology and future outlook.

BACKGROUND: Helminths are endemic in more than half of the world's countries, raising serious public health concerns. Accurate diagnosis of helminth infection is crucial to control strategies. Traditional parasitological methods, serological tests and PCR-based assays are the major means of the diagnosis of helminth infection, but they are time-consuming and/or expensive, and sometimes provide inaccurate results. Loop mediated isothermal amplification (LAMP) assay, a sensitive, simple and rapid method was therefore developed for detection of helminths. This study aims to discuss the current status of application of LAMP on helminths detection and to make a comprehensive evaluation about this updated technology and its future outlook by comparing with several other diagnostic methods. MAIN BODY: This review summarizes LAMP assay applied for helminth detection and helminthiasis surveillance. The basic principle of LAMP is introduced to help better understand its characteristics and each reported assay is assessed mainly based on its detection sensitivity, specificity and limitations, in comparison with other common diagnostic tests. Moreover, we discuss the limitations of the assays so as to clarify some potential ways of improvement. CONCLUSIONS: Here, we summarize and discuss the advantages, disadvantages and promising future of LAMP in heliminth detection, which is expected to help update current knowledge and future perspectives of LAMP in highly sensitive and specific diagnosis and surveillance of helminthiasis and other parasitic diseases, and can contribute to the elimination of the diseases from endemic areas.

Zoonotic parasites carried by invasive alien species in China.

BACKGROUND: The invasive alien species may lead to great environmental and economic crisis due to its strong capability of occupying the biological niche of native species and altering the ecosystem of the invaded area. However, its potential to serve as the vectors of some specific zoonotic pathogens, especially parasites, has been neglected. Thus, the damage that it may cause has been hugely underestimated in this aspect, which is actually an important public health problem. This paper aims to discuss the current status of zoonotic parasites carried by invasive alien species in China. MAIN BODY: This review summarizes the reported zoonotic parasites carried by invasive alien species in China based on the Database of Invasive Alien Species in China. We summarize their prevalence, threat to human health, related reported cases, and the roles of invasive alien species in the life cycle of these parasites, and the invasion history of some invasive alien species. Furthermore, we sum up the current state of prevention and control of invasive alien species in China, and discuss about the urgency and several feasible strategies for the prevention and control of these zoonoses under the background of booming international communications and inevitable globalization. CONCLUSIONS: Information of the zoonotic parasites carried by invasive alien species neither in China or worldwide, especially related case reports, is limited due to a long-time neglection and lack of monitoring. The underestimation of their damage requires more attention to the monitoring and control and compulsory measures should be taken to control the invasive alien species carrying zoonotic parasites.

Snail-borne parasitic diseases: an update on global epidemiological distribution, transmission interruption and control methods.

BACKGROUND: Snail-borne parasitic diseases, such as angiostrongyliasis, clonorchiasis, fascioliasis, fasciolopsiasis, opisthorchiasis, paragonimiasis and schistosomiasis, pose risks to human health and cause major socioeconomic problems in many tropical and sub-tropical countries. In this review we summarize the core roles of snails in the life cycles of the parasites they host, their clinical manifestations and disease distributions, as well as snail control methods. MAIN BODY: Snails have four roles in the life cycles of the parasites they host: as an intermediate host infected by the first-stage larvae, as the only intermediate host infected by miracidia, as the first intermediate host that ingests the parasite eggs are ingested, and as the first intermediate host penetrated by miracidia with or without the second intermediate host being an aquatic animal. Snail-borne parasitic diseases target many organs, such as the lungs, liver, biliary tract, intestines, brain and kidneys, leading to overactive immune responses, cancers, organ failure, infertility and even death. Developing countries in Africa, Asia and Latin America have the highest incidences of these diseases, while some endemic parasites have developed into worldwide epidemics through the global spread of snails. Physical, chemical and biological methods have been introduced to control the host snail populations to prevent disease. CONCLUSIONS: In this review, we summarize the roles of snails in the life cycles of the parasites they host, the worldwide distribution of parasite-transmitting snails, the epidemiology and pathogenesis of snail-transmitted parasitic diseases, and the existing snail control measures, which will contribute to further understanding the snail-parasite relationship and new strategies for controlling snail-borne parasitic diseases.

Soluble antigens from the neurotropic pathogen Angiostrongylus cantonensis directly induce thymus atrophy in a mouse model.

The nematode Angiostrongylus cantonensis (A.C.) is a neurotropic pathogen; stage-III larva invade the human (non-permissive host) central nervous system (CNS) to cause eosinophilic meningitis or meningoencephalitis accompanied by immunosuppression. In an A.C.-infectedmouse (another non-permissive host) model, CNS damage-associated T cell immune deficiency and severe inflammation were proposed to result from activation of the hypothalamic-pituitary-adrenal (HPA) axis. However, glucocorticoids are anti-inflammatory agents. Additionally, while defects in thymic stromal/epithelial cells (TECs) are the major reason for thymic atrophy, TECs do not express the glucocorticoid receptor. Therefore, activation of the HPA axis cannot fully explain the thymic atrophy and inflammation. Using an A.C.-infected mouse model, we found that A.C.-infected mice developed severe thymic atrophy with dramatic impairments in thymocytes and TECs, particularly cortical TECs, which harbor CD4+CD8+ double-positive thymocytes. The impairments resulted from soluble antigens (sAgs) from A.C. in the thymuses of infected mice, as intrathymic injection of these sAgs into live mice and the addition of these sAgs to thymic cell culture resulted in thymic atrophy and cellular apoptosis, respectively. Therefore, in addition to an indirect effect on thymocytes through the HPA axis, our study reveals a novel mechanism by which A.C. infection in non-permissive hosts directly induces defects in both thymocytes and TECs via soluble antigens.

A case report: A rare case of infant gastrointestinal canthariasis caused by larvae of Lasioderma serricorne (Fabricius, 1792) (Coleoptera: Anobiidae).

BACKGROUND: Canthariasis is a disease of humans caused by the infestation of beetle larvae. It is the second important insectal disease after myiasis. Several species of beetles are reported to cause the disease in gastrointestinal tract, urogenital system, nasal sinuses, ears and faces of mammals. The cigarette beetle Lasioderma serricorne is a widespread and destructive pest that usually feeds on tobacco, tea, beans, cereal grains, and animal and plant specimen. While there was no previous evidence of human infestation by this worm, we report the first case of L. serricorne infestation in a baby girl in China. CASE PRESENTATION: Here the case, an eight-month-old baby girl with irritable feeling, rubbing eyes, history of contact with mud and eating oranges twice during five days before attendance, and having "worms" in her stool was admitted to the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. The clinical examination revealed that the pulse rate, blood pressure and temperature were regular, and the examination of the head, neck, and chest were unremarkable. The stool specimens containing "worms" were sent to the Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University. The worms were recovered, studied morphologically using naked eyes and anatomical lens, PCR analyzed targeting cytochrome oxidase subunit 1 (COX1) and 18S rRNA genes, examined by sequence analyses of the PCR products and finally classified by phylogenetic analysis to identify their species. Based on the findings, the worms were diagnosed as the larvae of L. serricorne. CONCLUSION: This report implies that the baby had an infestation with the larvae of L. serricorne in the gastrointestine. During contact with mud or eating oranges by the girl, worm eggs were swallowed into the stomach and resisted gastric acid digestion which eventually hatched into larvae and caused canthariasis. The 8 months girl had underdeveloped immune system which might facilitate the disease. This report implicates that L. serricorne can infest human accidentally and cause canthariasis that may lead to severe damage to infant and older patient upon involvement of important organs of the body. The patients once diagnosed having canthariasis should be treated in time.

Nucleic acid detection in the diagnosis and prevention of schistosomiasis.

Schistosomiasis is an important zoonotic parasitic disease that causes serious harms to humans and animals. Surveillance and diagnosis play key roles in schistosomiasis control, however, current techniques for surveillance and diagnosis of the disease have limitations. As genome data for parasites are increasing, novel techniques for detection incorporating nucleotide sequences are receiving widespread attention. These sensitive, specific, and rapid detection methods are particularly important in the diagnosis of low-grade and early infections, and may prove to have clinical significance. This paper reviews the progress of nucleic acid detection in the diagnosis and prevention of schistosomiasis, including such aspects as the selection of target genes, and development and application of nucleic acid detection methods.

The expression of molecule CD28 and CD38 on CD4⁺/CD8⁺ T lymphocytes in thymus and spleen elicited by Schistosoma japonicum infection in mice model.

Schistosomiasis caused by human schistosomes such as Schistosoma japonicum (S. japonicum) is considered as an immune-related disease. It was demonstrated that specific cytokine antibodies' response elicited by S. japonicum infection was gradually downregulated with the progress of the disease, resulting in a Th1/Th2 polarization and suppression of immune response. CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival, and CD38 is an activating marker of T lymphocyte with high expression in many acute or chronic infections. The immune signature of CD28null T cells in the peripheral circulation associates with chronic inflammation in many diseases, such as HIV and CMV infection. In the thymus, CD28 expression on developing thymocytes appears to play a role for their selection, and it synergizes with CD38 to induce apoptosis of DP (double-positive) thymocytes. Few reports about CD28 and CD38 have been published in schistosomiasis. Here, we investigated the dynamic patterns of the expression of molecules CD28 and CD38 on CD4(+)/CD8(+) T lymphocytes of the thymus and spleen in mice model with S. japonicum infection. Our data indicated that at an early period of infection, the frequency of CD8(+)CD28(-) T cell in the spleen decreased significantly, but higher at chronic infection than that in control. However, it demonstrated an increasing trend in the thymus with the progression of infection. The frequency of CD4(+)CD28(-) T cells increased from acute infection in the thymus, while from chronic infection in the spleen. The expression of CD38 on CD8(+) T cells began to increase at 4 weeks post infection both in the thymus and spleen; its elevated expression on CD4(+) T cells emerged at 6 weeks post infection in the thymus and at 10 weeks post infection in the spleen. Praziquantel (PZQ) treatment could partially restore the frequency of CD28(+) T cell of CD4(+) T cells and CD38(+) T cell of CD8(+)/CD4(+) T cells in the spleen and CD38(+) T cell in the thymus. We hypothesized that the reactivation of S. japonicum infection may trigger expansion of CD28(-) T cells and hence mediate systemic inflammation. We speculated that CD8(+)CD28(-) T cell might be involved in immune modulation and CD8(+)CD28(-) T cell may be a crucial part in pathogenesis, which can provide further knowledge of the sophisticated mechanism of immuno-downregulation in schistosomiasis and potential treatment target.

Microglia activation: one of the checkpoints in the CNS inflammation caused by Angiostrongylus cantonensis infection in rodent model.

Angiostrongylus cantonensis (A. cantonensis) is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats; humans are non-permissive hosts like the mice. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningo-encephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of hosts to parasite during A. cantonensis invasion and development. To further understand the reasons why mice and rats attain different outcomes in A. cantonensis infection, we used the HE staining to observe the pathological changes of infected mice and rats. In addition, we measured mRNA levels of some cytokines (IL-5, IL-6, IL-13, Eotaxin, IL-4, IL-10, TGF-ß, IFN-γ, IL-17A, TNF-α, IL-1ß, and iNOS) in brain tissues of mice and rats by real-time PCR. The result showed that brain inflammation in mice was more serious than in rats. Meanwhile, mRNA expression levels of IL-6, IL-1ß, TNF-α, and iNOS increased after mice were infected. In contrast, mRNA levels of these cytokines in rats brain tissues decreased at post- infection 21 days. These cytokines mostly were secreted by activated microglia in central nervous system. Microglia of mice and rats were showed by Iba-1 (microglia marker) staining. In micee brains, microglia got together and had more significant activation than in rats brains. The results demonstrate that mice and rats have different CNS inflammation after infection by A. cantonensis, and it is in line with other researchers' reported findings. In conclusion, it is suggested that microglia activation is probably to be one of the most important factors in angiostrongyliasis from our study.

The mRNA level of the galectin-10 of Angiostrongylus cantonensis induced by reactive oxygen stress.

Galectin plays an important role in host-parasite interactions. In this study, we identified a novel gene encoding galectin-10 (AcGal-10) from the cDNA library of Angiostrongylus cantonensis and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcGal-10 is related to other galectin family members with the conserved loci (H(84)-D(86)-R(88)-V(96)-N(98)-W(105)-E(108)-R(110)). The mRNA level of AcGal-10 was expressed in reactive oxygen stress radicals. We have identified two proteins of A. cantonensis galectin-10 gene, one of which was reported (AcGAL10-W) and the others is AcGAL-10-M. In addition, recombinant AcGal-10 (rAcGal-10) was constructed into the pGEX-4T-1 plasmid, purified, and finally confirmed by SDS-PAGE and LC-MS. Hemagglutination assay showed that the minimum concentration of rAcGAL10-W and rAcGAL10-M required for the hemagglutination of BALB/c mice erythrocyte was 25 µg/mL, and the carbohydrate-binding ability showed no difference between rAcGAL10-W and rAcGAL10-M. The mRNA levels of AcGal-10 were indeed expressed higher after stimulation with H(2)O(2) and recombinant A. cantonensis galectin-10. A mutation of AcGal-10 was also found, but there was no significant difference compared with the wild type. Furthermore, we also confirmed that recombinant AcGal-10 plays a role in the activation of the microglia. In conclusion, the report here showed that AcGal-10 may be an important molecule related to infection of A. cantonensis.

[Skin inflammation responses induced by heat shock protein 70 derived from Schistosoma japonicum in BALB/c mice].

OBJECTIVE: To study the skin inflammation responses induced by heat shock protein 70 (rSj648/hsp70) from Schistosoma japonicum in BALB/c mice. METHODS: BALB/c mice were injected with 20 microg of LPS in abdominal skin or 100 microg of rSj648/hsp70, meanwhile, the PBS-treated group was set as blank. On days 1, 2, 4, 7 after the immunization, dynamic changes of inflammation were observed by staining with HE; and the IFN-gamma mRNA expression was detected by real-time PCR. Results On day 1, the inflammation of the skins derived from mice injected with LPS was obviously, and then fell off gradually after day 1. Compared with the LPS-treated group, the inflammation responses induced by Sj648/hsp70 were longer and more intensive until until day 7. CONCLUSIONS: Heat shock protein 70 (rSj648/hsp70) induces high protection against schistosome-infection contributing to predominant Th1 reaction and the correlation with high expression of IFN-gamma.

Cloning and characterization of a novel gene encoding 16 kDa protein (Ac16) from Angiostrongylus cantonensis.

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis or meningoencephalitis. A novel gene (AC16) was isolated from a cDNA library of A. cantonensis fourth-stage larvae. The putative 16-kDa protein has 149 amino acids and is homologous to an immunodominant hypodermal antigen (IHA16) from Ancylostoma caninum (identities = 57%). In this paper, we cloned the gene and purified the recombinant Ac16 (rAC16) protein. Real-time quantitative PCR revealed that Ac16 was expressed significantly higher in the fourth-stage larvae and adult worms derived from rats than that in the fourth-stage larvae derived from mice. Moreover, sera from rat (permissive host) infected with A. cantonensis detected Ac16 by Western blot, while sera from infected mouse (non-permissive host) could not. The results implied that Ac16 was related to the parasitic adaptation of A. cantonensis in different hosts and non-permissive host mouse had no circulating antibody to the antigen Ac16 from A. cantonensis and thus might contribute to understanding the mechanism of parasite immune evasion. Furthermore, we evaluated the ability of Ac16 antibody diagnosing A. cantonensis infection by an indirect enzyme-linked immunosorbent assay. The results showed that the Ac16 antibody had a 79.17% sensitivity to rAC16 and 83.33% to crude adult worm antigens (CA) (P > 0.05), while the specificity to rAC16 and to CA were 95.89% and 86.30% respectively (P < 0.05), thus implying that rAc16 may constitute a putative serodiagnostic antigen for Angiostrongyliasis cantonensis.

[Cloning, expression and immunologic identification of C31B8.8 gene of Caenorhabditis elegans].

OBJECTIVE: To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans, and study the immunological characteristic of the recombinant protein. METHODS: Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with (His) 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blotting 10 BALB/c mice were randomly divided into C31B.8-immunized group and PBS + adjuvant group Mice in C31B8.8-immunized group were immunized with 40 microg of purified C31B8.8 antigen formulated in Freund's adjuvant Mice in PBS + adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonensis fourth-stage larvae were identified by Western blotting. RESULTS: The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recombinant C31B8.8 protein was the target protein. Compared with PBS + adjuvant group, mice immunized with purified protein C31B8.8 produced higher level of IgG. The anti-C31B8.8 serum recognized recombinant C31B8.8 protein, and reacted with soluble antigens of A. cantonensis fourth-stage larvae. CONCLUSION: C elegans C31B8 gene shows certain immunogenicity and immunoreactivity, and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8 serum.

Intranasal immunization with autolysin (LytA) in mice model induced protection against five prevalent Streptococcus pneumoniae serotypes in China.

In order to evaluate immunogenicity and protective efficacy of LytA from Streptococcus pneumoniae, we subcloned the full-length lytA-encoded autolysin (LytA) from 5 major pathogenic serotype isolates in China and obtained purified rLytA. Bioinformatics analysis showed that sequences of LytA were highly conserved in all strains we used in this work, and western blot analysis demonstrated that rLytAs from heterogeneous serotypes were cross-recognized by serum of mice infected with 23F strain SH137. Mice were intranasally immunized with purified rLytA, and serum anti-rLytA IgG, IgA and secretory IgA were elicited. More importantly, rLytA intranasal-immunized mice showed a significantly higher survival rate and lower bacterial carriage in response to infection by Streptococcus pneumoniae. The fact that mice immunized with rLytA from strain SH137 also had a higher survival rate after intraperitoneal injection of other four serotype strains of living S. pneumoniae suggested that it possessed cross-protection effect. Our study revealed that intranasal immunization with rLytA may protect mice against mucosal and systemic pneumococcal infection; hence, it was an attractive vaccine candidate.

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.

Efficacy of combined treatment with albendazole and baicalein against eosinophilic meningitis induced by Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis infection causes eosinophilic meningitis in humans. Baicalein is a flavonoid originally isolated from the roots of Scutellaria baicalensis Georgi. In this study we evaluated the efficacy of the combination of albendazole and baicalein for treating eosinophilic meningitis in BALB/c mice. Therapeutic efficacy included the survival time, body weight, neurological function, leucocyte and eosinophil counts, eotaxin concentration, matrix metalloproteinase-9 (MMP-9) activity, larval recovery and histopathological examination. The results showed that the combination of albendazole and baicalein was more effective than either drug administered singly. Combination therapy increased the survival time, decreased body weight loss, neurological dysfunction, leucocyte response, eotaxin concentration and MMP-9 activity. Our results suggest that the combination of albendazole and baicalein may exhibit synergistic beneficial effects in the treatment of eosinophilic meningitis induced by A. cantonensis.