Jinlin Zhou

Babesia parasites develop and are transmitted by the non-vector soft tick Ornithodoros moubata (Acari: Argasidae).

Ornithodoros moubata ticks were fed on blood infected with Babesia equi. However, the parasites were quickly cleared as evidenced by the disappearance of B. equi-specific ribosomal RNA from the ticks. We hypothesized that if the Babesia parasite can escape midgut-associated barriers a non-vector tick can become infected with Babesia. To test this hypothesis, B. equi parasite-infected blood from in vitro culture was injected into the haemocoel of ticks. B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs. Babesia-free dogs were infested with O. moubata ticks that were infected by inoculation with B. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP of B. gibsoni increased gradually. These results indicate that O. moubata may be a useful vector model for Babesia parasites and also a very important tool for studies on tick immunity against Babesia parasites and tick-Babesia interactions.

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis.

Two cDNAs encoding defensin-like antimicrobial peptides were cloned and sequenced from the tick Haemaphysalis longicornis. The full-length cDNA of Hlgut-defensin (H. longicornis midgut defensin) is 333bp, encoding an expected protein with 73 amino acids. The full-length cDNA of Hlsal-defensin (H. longicornis salivary gland defensin) is 382bp, encoding an expected protein with 81 amino acids. The antibacterial activities of the synthetic peptides based on the Hlgut-defensin and Hlsal-defensin sequences were tested against a variety of Gram-positive and Gram-negative bacteria. Using real-time PCR, the tissue-specific expression of two defensin-like peptides were determined and it was also found that the gene transcripts of Hlgut-defensin and Hlsal-defensin were significantly induced by a lipopolysaccharide (LPS) injection.

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.

A heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, induced protective immunity against Toxoplasma gondii infection in mice.

SUMMARYThe dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-gamma) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.

RNA interference of cytosolic leucine aminopeptidase reduces fecundity in the hard tick, Haemaphysalis longicornis

Ticks are effective vectors of pathogens because of their blood feeding and high fecundity. This high fecundity is related to the size of the blood meal. Therefore, knowledge of how blood proteins are degraded and converted to proteins, including yolk protein, is important for the development of ways to inhibit the utilization of blood proteins by ticks. RNA interference (RNAi) is becoming a powerful post-transcriptional gene silencing technique that provides insight into gene function. We constructed a double-stranded RNA (dsRNA) based on a previously cloned Haemaphysalis longicornis leucine aminopeptidase (HlLAP) gene to reevaluate the biological role in tick blood digestion. Gene specific transcriptional, translational, and functional disruptions were achieved by the introduction of dsRNA into the ticks. Significantly delayed onset of egg-laying and reduced egg oviposition resulted from the RNAi for the HlLAP gene. These results suggest that HlLAP actually works as a blood digestive enzyme and affects tick fecundity via unknown mechanisms. The reduction of egg oviposition may be caused by a decrease in nutrients, especially free amino acids generated by HlLAP, from the blood meal. This is the first report of an impact on tick reproduction caused by gene silencing of a blood digestion-related molecule.

Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice.

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.

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.

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite–host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.