An unconventional SNARE complex mediates exocytosis at the plasma membrane and vesicular fusion at the apical annuli in Toxoplasma gondii.

Exocytosis is a key active process in cells by which proteins are released in bulk via the fusion of exocytic vesicles with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-mediated vesicle fusion with the plasma membrane is essential in most exocytotic pathways. In mammalian cells, the vesicular fusion step of exocytosis is normally mediated by Syntaxin-1 (Stx1) and SNAP25 family proteins (SNAP25 and SNAP23). However, in Toxoplasma gondii, a model organism of Apicomplexa, the only SNAP25 family protein, with a SNAP29-like molecular structure, is involved in vesicular fusion at the apicoplast. Here, we reveal that an unconventional SNARE complex comprising TgStx1, TgStx20, and TgStx21 mediates vesicular fusion at the plasma membrane. This complex is essential for the exocytosis of surface proteins and vesicular fusion at the apical annuli in T. gondii.

African Swine Fever Virus HLJ/18 CD2v Suppresses Type I IFN Production and IFN-Stimulated Genes Expression through Negatively Regulating cGMP-AMP Synthase-STING and IFN Signaling Pathways.

African swine fever is a fatal infectious disease caused by African swine fever virus (ASFV). The high mortality caused by this infectious disease is a significant challenge to the swine industry worldwide. ASFV virulence is related to its ability to antagonize IFN response, yet the mechanism of antagonism is not understood. Recently, a less virulent recombinant virus has emerged that has a EP402R gene deletion within the parental ASFV HLJ/18 (ASFV-ΔEP402R) strain. EP402R gene encodes CD2v. Hence we hypothesized that ASFV uses CD2v protein to evade type I IFN-mediated innate immune response. We found that ASFV-ΔEP402R infection induced higher type I IFN response and increased the expression of IFN-stimulated genes in porcine alveolar macrophages when compared with parental ASFV HLJ/18. Consistent with these results, CD2v overexpression inhibited type I IFN production and IFN-stimulated gene expression. Mechanistically, CD2v, by interacting with the transmembrane domain of stimulator of IFN genes (STING), prevented the transport of STING to the Golgi apparatus, and thereby inhibited the cGMP-AMP synthase-STING signaling pathway. Furthermore, ASFV CD2v disrupted IFNAR1-TYK2 and IFNAR2-JAK1 interactions, and thereby inhibited JAK-STAT activation by IFN-α. In vivo, specific pathogen-free pigs infected with the mutant ASFV-ΔEP402R strain survived better than animals infected with the parental ASFV HLJ/18 strain. Consistent with this finding, IFN-ß protein levels in the peripheral blood of ASFV-ΔEP402R-challenged pigs were significantly higher than in the blood of ASFV HLJ/18-challenged pigs. Taken together, our findings suggest a molecular mechanism in which CD2v inhibits cGMP-AMP synthase-STING and IFN signaling pathways to evade the innate immune response rendering ASFV infection fatal in pigs.

The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii.

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.

GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon.

Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

Localization and enzyme kinetics of aminopeptidase N3 from Toxoplasma gondii.

Aminopeptidase N is an important metalloenzyme from the M1 zinc metallopeptidase family, which is present in numerous apicomplexan parasites, including Plasmodium, Eimeria, and Cryptosporidium. Aminopeptidase N is a potential drug target, and hence, its properties have been widely investigated. In the current study, the cellular localization and enzyme characteristics of Toxoplasma gondii aminopeptidase N3 (TgAPN3) were evaluated in vitro. Cellular localization analysis revealed that TgAPN3 and GRA protein were co-located in the organelle and parasitophorous vacuole of T. gondii. The secretion assay showed that TgAPN3 could be co-secreted from the tachyzoites with GRA protein. A functional recombinant Toxoplasma aminopeptidase N3 (rTgAPN3) was produced in Escherichia coli. The enzyme activity was first determined using a fluorogenic H-Ala-MCA substrate. Some activity of rTgAPN3 was observed between pH 3.0 and 8.0, with a peak at pH 7.0. The activity was significantly enhanced in the presence of Co2+ ions. Substrate specificity of rTgAPN3 was then evaluated. The enzyme showed a preference for substrates containing N-terminal Ala residues, followed by Tyr and Cys. The rTgAPN3 activity was significantly inhibited by bestatin and phebestatin. In general, TgAPN3 was a structurally conserved member of the M1 family, although it also displayed unique biochemical characteristics. These results lay the foundation for a functional study of TgAPN3 and constitute its putative identification as a drug target.

Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

The Nrf2 pathway is required for intracellular replication of Toxoplasma gondii in activated macrophages.

Reactive oxygen species (ROS) produced by oxidases and nonenzymatic sources are important for host defence against intracellular pathogens. In this study, we knocked out the Nrf2 gene in RAW264.7 cells using the CRISPR/Cas9 system and investigated the antioxidant effects of the Nrf2 pathway in the cells stimulated by IFN-γ and TNF-α. The results indicated that the Nrf2 signalling pathway is necessary for maintaining redox homeostasis in activated RAW264.7 cells. Inactivation of Nrf2 impaired parasite growth. We also found that p62 contributes to Nrf2-mediated pathways involved in T gondii infection. These findings suggest that the Nrf2/Keap1 pathway may be targeted to prevent and treat toxoplasmosis.

The moving junction protein RON4, although not critical, facilitates host cell invasion and stabilizes MJ members.

Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa. To facilitate high-efficiency invasion of host cells, T. gondii secretes various proteins related to the moving junction (MJ) complex from rhoptries and micronemes into the interface between the parasite and host. AMA1/RON2/4/5/8 is an important MJ complex, but its mechanism of assembly remains unclear. In this study, we used the CRISPR-Cas9 system to generate a derivative of T. gondii strain RH with a null mutation in TgRON4, thought to be an essential MJ component. Deficiency of TgRON4 moderately decreased invasion ability relative to that of the wild-type parasite. In addition, expression of the endogenous N-terminal fragment of RON5 decreased in the mutant. Together, the results improve our understanding of the assembly mechanism of the MJ complex of T. gondii and raise the possibility of developing new therapeutic drugs that target this complex.

Effects of dihydroorotate dehydrogenase (DHODH) inhibitors on the growth of Theileria equi and Babesia caballi in vitro.

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC50) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 µM, respectively, whereas the IC50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 µM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP.

Expression of truncated Babesia microti apical membrane protein 1 and rhoptry neck protein 2 and evaluation of their protective efficacy.

In this study, we evaluated the protective effect of recombinant Babesia microti apical membrane protein 1 (rBmAMA1) and rhoptry neck protein 2 (rBmRON2) against B. microti infection using a hamster model. The genes encoding the predicted domains I and II of BmAMA1 and the gene encoding the predicted transmembrane regions 2 and 3 of BmRON2 were expressed as His fusion recombinant proteins in Escherichia coli. Three groups with 5 hamsters in each group were immunized with rBmAMA1, rBmRON2 and rBmAMA1+rBmRON2, then challenged with B. microti. The result showed that only the group immunized with rBmAMA1+rBmRON2 exhibited limited protection against B. microti challenge infection, characterized by significant decreased of parasitemia and higher hematocrit values from day 6-10 post challenge infection. However, there was no significant difference in the groups immunized with rBmAMA1 or rBmRON2 alone. The absence of a significant difference in the total amount of antibodies against rBmAMA1 and rBmRON2 between the group immunized with single and combined proteins. This result suggests that the protection cannot be solely attributed to the quantity of antibodies produced, but also to their ability to target important epitopes from both antigens. These results suggest that combined immunization with rBmAMA1 and rBmRON2 is a promising strategy against B. microti.

DB-AT: a 2015 update to the Full-parasites database brings a multitude of new transcriptomic data for apicomplexan parasites.

The previous release of our Full-parasites database (http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909,150,388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT--DataBase of Apicomplexa Transcriptomes.

Molecular detection and genetic diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand.

Babesia spp., Theileria orientalis, and Anaplasma marginale are significant tick-borne pathogens that affect the health and productivity of cattle in tropical and subtropical areas. In this study, we used PCR to detect the presence of Babesia bovis, Babesia bigemina, and T. orientalis in 279 beef cattle from Western Thailand and A. marginale in 608 beef cattle from the north, northeastern, and western regions. The PCRs were performed using species-specific primers based on the B. bovis spherical body protein 2 (BboSBP2), B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. orientalis major piroplasm surface protein (ToMPSP), and A. marginale major surface protein 4 (AmMSP4) genes. To determine the genetic diversity of the above parasites, amplicons of B. bovis and B. bigemina ITS1-5.8s rRNA gene-ITS2 regions (B. bovis ITS, B. bigemina ITS), ToMPSP, and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the prevalence of B. bovis, B. bigemina, T. orientalis, and A. marginale in the Western region was 11.1, 12.5, 7.8, and 39.1 %, respectively. Coinfections of two or three parasites were observed in 17.9 % of the animals sampled. The study revealed that the prevalence of A. marginale in the western region was higher than in the north and northeastern regions (7 %). Sequence analysis showed the BboSBP2 gene to be more conserved than B. bovis ITS in the different isolates and, similarly, the BbiRAP-1a was more conserved than B. bigemina ITS. In the phylogenetic analysis, T. orientalis MPSP sequences were classified into types 3, 5, and 7 as previously reported. A. marginale MSP4 gene sequences shared high identity and similarity with each other and clustered with isolates from other countries. This study provides information on the prevalence and genetic diversity of tick-borne pathogens in beef cattle and highlights the need for effective strategies to control these pathogens in Thailand.

Primary Babesia rodhaini infection followed by recovery confers protective immunity against B. rodhaini reinfection and Babesia microti challenge infection in mice.

In the present study, we investigated the protective immunity against challenge infections with Babesia rodhaini and Babesia microti in the mice recovered from B. rodhaini infection. Six groups with 5 test mice in each group were used in this study, and were intraperitoneally immunized with alive and dead B. rodhaini. The challenge infections with B. rodhaini or B. microti were performed using different time courses. Our results showed that the mice recovered from primary B. rodhaini infection exhibited low parasitemia and no mortalities after the challenge infections, whereas mock mice which had received no primary infection showed a rapid increase of parasitemia and died within 7 days after the challenge with B. rodhaini. Mice immunized with dead B. rodhaini were not protected against either B. rodhaini or B. microti challenge infections, although high titers of antibody response were induced. These results indicate that only mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini or B. microti challenge infection. Moreover, the test mice produced high levels of antibody response and low levels of cytokines (INF-γ, IL-4, IL-12, IL-10) against B. rodhaini or B. microti after challenge infection. Mock mice, however, showed rapid increases of these cytokines, which means disordered cytokines secretion occurred during the acute stage of challenge infection. The above results proved that mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini and B. microti infections.

Macrophages are the determinant of resistance to and outcome of nonlethal Babesia microti infection in mice.

In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ(-/-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis.

Boosting PRRSV-Specific Cellular Immunity: The Immunological Profiling of an Fc-Fused Multi-CTL Epitope Vaccine in Mice.

The continuously evolving PRRSV has been plaguing pig farms worldwide for over 30 years, with conventional vaccines suffering from insufficient protection and biosecurity risks. To address these challenges, we identified 10 PRRSV-specific CTL epitopes through enzyme-linked immunospot assay (ELISPOT) and constructed a multi-epitope peptide (PTE) by linking them in tandem. This PTE was then fused with a modified porcine Fc molecule to create the recombinant protein pFc-PTE. Our findings indicate that pFc-PTE effectively stimulates PRRSV-infected specific splenic lymphocytes to secrete high levels of interferon-gamma (IFN-γ) and is predicted to be non-toxic and non-allergenic. Compared to PTE alone, pFc-PTE not only induced a comparable cellular immune response in mice but also extended the duration of the immune response to at least 10 weeks post-immunization. Additionally, pFc-PTE predominantly induced a Th1 immune response, suggesting its potential advantage in enhancing cellular immunity. Consequently, pFc-PTE holds promise as a novel, safe, and potent candidate vaccine for PRRSV and may also provide new perspectives for vaccine design against other viral diseases.

Duck Tembusu virus infection activates the MKK3/6-p38 MAPK signaling pathway to promote virus replication.

Duck Tembusu virus (DTMUV) infection poses a serious threat to ducks, chickens, and geese, causing a range of detrimental effects, including reduced egg production, growth retardation, and even death. These consequences lead to substantial economic losses for the Chinese poultry industry. Although it is established that various viral infections can trigger activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, the precise role and mechanisms underlying p38 MAPK activation in DTMUV infection remain poorly understood. To address this knowledge gap, we conducted a study to investigate whether the replication of DTMUV necessitates the activation of p38 MAPK. We found that DTMUV infection stimulates activation of the MKK3/6-p38 MAPK pathway, and the activation of p38 MAPK increases with viral titer. Subsequently, the use of the small molecule inhibitor SB203580 significantly reduced DTMUV replication by inhibiting p38 MAPK activity. Furthermore, downregulation of p38 MAPK protein expression by siRNA also inhibited DTMUV replication, whereas transient transfection of p38 MAPK protein promoted DTMUV replication. Interestingly, we found that the DTMUV capsid protein activates p38 MAPK, and there is interaction between DTMUV capsid and p38 MAPK. Finally, we found that DTMUV infection induces elevated mRNA expression of IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-6, and IL-12, which is associated with p38 MAPK activity. These results indicated that virus hijacking of p38 activation is a crucial event for DTMUV replication, and that pharmacological blockade of p38 activation represents a potential anti-DTMUV strategy.

Identification and characterization of an interspersed repeat antigen of Babesia microti (BmIRA).

In this report, a novel gene encoding an interspersed repeat antigen from Babesia microti (BmIRA) was identified and described. The full-length cDNA containing an open reading frame of 1,947 bp was obtained by immunoscreening a B. microti cDNA expression library. The full-length of BmIRA gene was expressed as a GST fusion recombinant BmIRA (rBmIRA) in Escherichia coli. Sera of mice immunized with the rBmIRA detected a native parasite protein with a molecular mass of 76 kDa on Western blot analysis. The same protein was detected in the parasites by immunofluorescent antibody test (IFAT). An enzyme-linked immunosorbent assay (ELISA) using rBmIRA detected specific antibodies as early as 11 days post-infection in sera from a hamster experimentally infected with B. microti Gray stain (US type). Furthermore, a rapid immunochromatographic test (ICT) using rBmIRA detected specific antibodies in a hamster experimentally infected with B. microti from day 11 to at least day 180 post-infection. The results indicate the antibody response against the rBmIRA was maintained during the chronic stage of infection. On the other hand, an immunoprotective property of rBmIRA as a subunit vaccine was evaluated in hamsters against B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmIRA could be a useful serodiagnostic antigen for screening of B. microti infection.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Recombinant Pseudorabies Virus Usage in Vaccine Development against Swine Infectious Disease.

Pseudorabies virus (PRV) is the pathogen of pseudorabies (PR), which belongs to the alpha herpesvirus subfamily with a double stranded DNA genome encoding approximately 70 proteins. PRV has many non-essential regions for replication, has a strong capacity to accommodate foreign genes, and more areas for genetic modification. PRV is an ideal vaccine vector, and multivalent live virus-vectored vaccines can be developed using the gene-deleted PRV. The immune system continues to be stimulated by the gene-deleted PRVs and maintain a long immunity lasting more than 4 months. Here, we provide a brief overview of the biology of PRV, recombinant PRV construction methodology, the technology platform for efficiently constructing recombinant PRV, and the applications of recombinant PRV in vaccine development. This review summarizes the latest information on PRV usage in vaccine development against swine infectious diseases, and it offers novel perspectives for advancing preventive medicine through vaccinology.