Yongzhi Zhou

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.

Characterization of the anticoagulant protein Rhipilin-1 from the Rhipicephalus haemaphysaloides tick

To understand the molecular mechanism of tick blood feeding, an anticoagulant protein, Rhipilin-1, was identified in the tick Rhipicephalus haemaphysaloides. The cDNA sequence of Rhipilin-1 is 620bp, and it encodes a deduced 164 amino acid protein with a size of 18kDa. Bioinformatic analysis shows that Rhipilin-1 belongs to the Kunitz-type family of inhibitors, containing one Kunitz domain with high homology to the tissue factor pathway inhibitor (TFPI). The recombinant protein expressed in Escherichia coli delayed normal clotting of rabbit plasma both in the recalcification time (RT) and the activated partial thromboplastin time (APTT) tests. Using RT-PCR, mRNA transcripts of Rhipilin-1 were detected in fed but not in unfed ticks. Disruption of the Rhipilin-1 gene with RNAi led to a 52.7% decrease in the tick attachment rate 24h after introduction in the rabbit ears and a 21.9% decrease in the average engorged body weight of ticks. These results indicate that Rhipilin-1 is a novel anticoagulant protein involved in tick blood feeding with possible future application as a vaccine candidate. The discovery of Rhipilin-1 is the first report on anticoagulant genes in this species of tick.

Isolation and genotyping of Toxoplasma gondii from domestic rabbits in China to reveal the prevalence of type III strains.

In this study, Toxoplasma gondii antibodies in 77 free domestic rabbits from a rural area surrounding Shanghai, China were analyzed via ELISA, which identified 18 seropositive rabbits. One strain of T. gondii (designated SHR) was successfully isolated from one seropositive rabbit using a mouse bioassay. The isolated T. gondii killed all BALB/c mice inoculated with 10(4) tachyzoites, indicating its virulence in mice. Mn-PCR-RFLP analysis was used to type parasites recovered from cell cultures. Further analysis based on sequencing of a polymorphic intron revealed that the isolated strain contained a clonal type III genome, a rare finding in any host in China. This is the first reported isolation of T. gondii genotype III from rabbits in China. Our results suggested that type III strains are circulating in rabbits in China, which act as potential reservoirs of T. gondii transmission.

Isolation and characterization of two novel serpins from the tick Rhipicephalus haemaphysaloides.

Two novel serpins with anti-chymotrypsin activity, RHS-1 and RHS-2, were identified in the tick Rhipicephalus haemaphysaloides. The complementary cDNA sequence of RHS-1 was 1286 base pairs (bp) and encoded a deduced 403-amino acid protein with a signal peptide, whereas that of RHS-2 was 1682bp and encoded a deduced 380-amino acid protein with no signal peptide. Although both RHS-1 and RHS-2 exhibited high sequence similarities to known serpins from other ticks, the level of similarity at the amino acid level between the 2 serpins characterized here was only 32.5%. Salivary gland-specific expression of RHS-1 and midgut-specific expression of RHS-2 were found by Western blot using the relevant antiserum. We tested the ability of purified recombinant rRHS-1 and rRHS-2 to inhibit various serine proteases and found that both significantly inhibited chymotrypsin (95.6% and 94.2%, respectively). We further demonstrated that RHS-1, but not RHS-2 exhibited anticoagulation activity, based on activated partial thromboplastin time (APTT). Disruption of the genes encoding the 2 serpins with RNA interference (RNAi) led to a significant decrease in tick attachment and engorgement rates. These results indicate that RHS-1 and RHS-2 are 2 novel serpins with anti-chymotrypsin activity that are involved in blood feeding of R. haemaphysaloides.

CHARACTERIZATION OF A NEW KUNITZ-TYPE SERINE PROTEASE INHIBITOR FROM THE HARD TICK Rhipicephalus hemaphysaloides

A new Kunitz-type serine protease inhibitor, Rhipilin-2, was identified in the tick Rhipicephalus hemaphysaloides. The cDNA sequence of Rhipilin-2 is 693 bp, and it encodes a deduced 195 amino acid protein with a size of 22 kDa. Bioinformatic analysis shows that Rhipilin-2 belongs to the Kunitz-type family of inhibitors, containing one Kunitz domain with homology to the tissue factor pathway inhibitor. Using Real time polymerase chain reaction (Real time-PCR), Rhipilin-2 mRNA transcripts were detected in tick salivary glands and midgut. Blood feeding induced transcript expression. The recombinant protein was expressed in insect Sf9 cells and confirmed by immunofluorescence test and Western blot analysis with an anti-His antibody. The purified recombinant Rhipilin-2 inhibited serine protease trypsin and elastase, but not thrombin. The anticoagulant activity of Rhipilin-2 was shown by delaying normal clotting of rabbit plasma in the activated partial thromboplastin time tests. These results indicate that Rhipilin-2 is a novel Kunitz-type serine protease inhibitor involved in tick blood feeding.

Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides

The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animals body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastase-inhibitory activity and markedly inhibited the growth of gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides.

Seroprevalence survey of Babesia gibsoni infection and tick species in dogs in East China.

A seroprevalence survey of Babesia gibsoni infection in dogs in East China was conducted using an ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1170 dogs from East China were examined and the seroprevalence was 9.23%. The proportion of samples was 81.2%, 7.86% and 10.94% from pet, working and fighting dogs, respectively. The fighting dogs showed highest seroprevalence (39.8%) compared with working dogs (26.1%) and pet dogs (3.47%). These results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout East China. The dominant ticks collected from the dogs were identified as Rhipicephalus sanguineus (65.57%), Haemaphysalis longicornis (21.58%) and Rhipicephalus hemaphysaloides (10.7%). Besides adult, larval and nymph stages of ticks were also recorded on dogs. This is the first report of seroprevalence of canine B. gibsoni infection and tick species in dogs in China.

Determination of stage interconversion in vitro and in vivo by construction of transgenic Toxoplasma gondii that stably express stage-specific fluorescent proteins.

Detection of Toxoplasma gondii conversion from the tachyzoite stage to the bradyzoite stage in living brain tissue is difficult because the parasites are small and conversion and reactivation of the parasites are transient events. To better understand the mechanisms of T. gondii stage conversion between tachyzoites and bradyzoites, and to recognize stage conversion in an intermediate host, we constructed a transgenic cyst-forming strain (PLK) of T. gondii. The parasites stably expressed enhanced green fluorescence protein (EGFP) in the tachyzoite stage and red fluorescence protein (RFP) in the bradyzoite stage, under the control of the SAG1 and BAG1 promoters, respectively. The resulting transgenic parasite was designated as PLK/Bi. The PLK/Bi zoites expressed only green fluorescence in the tachyzoite stage and only red fluorescence in the bradyzoite stage in vitro and in vivo. Fluorescence analyses showed that recombinant GFP and RFP were located to the intracellular vacuolar spaces. In addition, an analysis of growth and culture conditions of transgenic T. gondii was performed in vitro and the virulence was evaluated in vivo. Our data suggested that the stage-specific fluorescence expression by PLK/Bi may be rationally designed for in vitro and in vivo studies on stage conversion and reactivation of T. gondii.

The immunomodulatory protein RH36 is relating to blood-feeding success and oviposition in hard ticks

An immunomodulatory protein designated RH36 was identified in the tick Rhipicephalus haemaphysaloides. The cDNA sequence of RH36 has 844bp and encodes a deduced protein with a predicted molecular weight of 24kDa. Bioinformatics analysis indicated that RH36 presented a degree of similarity of 34.36% with the immunomodulatory protein p36 from the tick Dermacentor andersoni. The recombinant RH36 (rRH36) expressed in Sf9 insect cells suppressed the T-lymphocyte mitogen-driven in vitro proliferation of splenocytes and the expression of several cytokines such as IL-2, IL-12, and TNF-α. Furthermore, the proliferation of splenocytes isolated from rRH36-inoculated mice was significantly lower than that in control mice, suggesting that rRH36 could directly suppress immune responses in vivo. In addition, microarray analysis of splenocytes indicated that the expression of several immunomodulatory genes was downregulated by rRH36. The silencing of the RH36 gene by RNAi led to a 37.5% decrease in the tick attachment rate 24h after placement into the rabbit ears, whereas vaccination with RH36 caused a 53.06% decrease in the tick engorgement rate. Unexpectedly, RNAi induced a significant decrease in the oviposition rate, ovary weight at day 12 after engorgement, and egg-hatching rate. The effects of RH36 on blood feeding and oviposition were further confirmed by vaccination tests using the recombinant protein. These results indicate that RH36 is a novel member of immunosuppressant proteins and affects tick blood feeding and oviposition.

Babesia microti Aldo-keto Reductase-Like Protein Involved in Antioxidant and Anti-parasite Response

The intraerythrocytic apicomplexan Babesia microti is the primary causative agent of human babesiosis, which is an infectious disease that occurs in various regions around the world. Although the aldo-keto reductases (AKRs) of this parasite have been sequenced and annotated, their biological properties remain unknown. AKRs are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. In the present study, we cloned the full-length cDNA of a B. microti aldo-keto reductase-like protein (BmAKR) and analyzed the deduced amino acid sequence of the BmAKR protein. This protein has a conserved AKR domain with an N-terminal signal sequence. Bmakr was upregulated on the 8th day after infection, whereas it was downregulated during the later stages. The recombinant protein of BmAKR was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli. Western blot analysis showed that the mouse anti-BmAKR antibody recognized native BmAKR from a parasite lysate. Immunofluorescence microscopy localized BmAKR to the cytoplasm of B. microti merozoites in mouse RBCs in this study. Bmakr expression was significantly upregulated in the presence of oxidant stress. Atovaquone, a known anti-babesiosis drug, and robenidine, a known anti-coccidiosis drug, induced upregulation of Bmakr mRNA, thereby suggesting that Bmakr may be involved in anti-parasite drug response.