Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Changes in TFG gene expression in bovine leucocytes transformed by Theileria annulata.

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

Assessment of probiotic adhesion and inhibitory effect on Escherichia coli and Salmonella adhesion.

In this study, we screened bacterial strains to identify specific probiotics to treat pig diarrhea caused by Escherichia coli or Salmonella. The potential probiotics were assayed for their survival in gastrointestinal solution, their antimicrobial activity, cell-surface properties, adhesion to Caco-2 cells, and inhibition of pathogen adhesion. Nine out of the 20 strains tested showed high tolerance of a simulated gastrointestinal environment and six strains exerted antagonistic effects against enterohemorrhagic E. coli (EHEC) O157:H7 and Salmonella Typhimurium MQ. Lactobacillus johnsonii pDX1e exhibited a higher potent antibacterial activity. Four strains (pDX1a, pDX1e, pDX3a, and pDX5a) displayed auto-aggregation, hydrophobicity, and adhesion to Caco-2 cells similar to those of the reference strain Lacticaseibacillus rhamnosus GG (LGG). Enterococcus durans pDX5a showed the highest adhesion capacity (13.86%), followed by the reference strain LGG (11.20%). All the tested strains competitively suppressed the attachment of pathogens to Caco-2 cells (by 30.73-55.18%); L. johnsonii pDX1e and Ent. durans pDX5a significantly inhibited the adhesion of pathogens by substitution and exclusion, respectively. Therefore, pDX1e and pDX5a were selected as probiotic strains for further investigation and application.

A Novel miRNA-hlo-miR-2-Serves as a Regulatory Factor That Controls Molting Events by Targeting CPR1 in Haemaphysalis longicornis Nymphs.

Successful completion of the molting process requires new epidermal growth and ecdysis of the old cuticle in Haemaphysalis longicornis (H. longicornis). MicroRNAs (miRNAs) participate in the development of organisms by inhibiting the expression of their target mRNAs. In this study, a novel tick-specific miRNA was identified and denoted hlo-miR-2 that serves as a novel regulator of molting events in H. longicornis nymphs by targeting a cuticular protein. The full length of this cuticular protein was first obtained and named it CPR1. A qRT-PCR analysis showed that hlo-miR-2 and CPR1 exhibit significant tissue and temporal specificity and that their transcription levels are negatively correlated during the molting process. CPR1, as a direct target of hlo-miR-2, was identified by a luciferase reporter assay in vitro. Agomir treatment indicated that the overexpression of hlo-miR-2 significantly reduced the protein expression level of CPR1, decreased the molting rate and delayed the molting time point in H. longicornis nymphs. RNA interference (RNAi) experiments demonstrated that CPR1 was significantly associated with the molting process in H. longicornis nymphs. Phenotypic rescue experiments convincingly showed that hlo-miR-2 participated in molting events by targeting CPR1 in H. longicornis nymphs. In summary, we present evidence demonstrating that miRNAs constitute a novel important regulator of molting events in addition to hormones. The described functional evidence implicating CPR1 in molting events contributes to an improved understanding of the distinct functions of the CPR family in ticks and will aid the development of a promising application of cuticular protein RNAi in tick control.

Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau.

Anaplasma species are tick-transmitted obligate intracellular bacteria that infect many wild and domestic animals and humans. The prevalence of Anaplasma spp. in ixodid ticks of Qinghai Province is poorly understood. In this study, a total of 1104 questing adult ticks were investigated for the infection of Anaplasma species. As a result, we demonstrated the total infection rates of 3.1, 11.1, 5.6, and 4.5% for A. phagocytophilum, A. bovis, A. ovis and A. capra, respectively. All of the tick samples were negative for A. marginale. The positive rates of A. phagocytophilum, A. ovis and A. capra in different tick species were significantly different. The positive rates of A. capra and A. bovis in the male ticks were significantly higher than that in the female ticks. Sequence analysis of A. ovis showed 99.5-100% identity to the previous reported isolates. The sequences of A. phagocytophilum had 100% identity to strains Ap-SHX21, JC3-3 and ZAM dog-181 from sheep, Mongolian gazelles, and dogs. Two genotypes of A. capra were found based on 16S rRNA, citrate synthase (gltA) gene and heat shock protein (groEL) gene analysis. In conclusion, A. bovis, A. ovis, A. phagocytophilum, and A. capra were present in the ticks in Qinghai Province. Anaplasma infection is associated with tick species, gender and distribution. These data will be helpful for understanding prevalence status of Anaplasma infections in ticks in Qinghai-Tibet Plateau.

Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii.

BACKGROUND: Although sexual reproduction of the parasite Toxoplasma gondii exclusively occurs in the cat intestine, knowledge about the alteration of gene expression in the intestine of cats infected with T. gondii is still limited. Here, we investigated the temporal transcriptional changes that occur in the cat intestine during T. gondii infection. METHODS: Cats were infected with 100 T. gondii cysts and their intestines were collected at 6, 12, 18, 24, 72 and 96 hours post-infection (hpi). RNA sequencing (RNA-Seq) Illumina technology was used to gain insight into the spectrum of genes that are differentially expressed due to infection. Quantitative RT-PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing. RESULTS: Our transcriptome analysis revealed 2363 DEGs that were clustered into six unique patterns of gene expression across all the time points after infection. Our analysis revealed 56, 184, 404, 508, 400 and 811 DEGs in infected intestines compared to uninfected controls at 6, 12, 18, 24, 72 and 96 hpi, respectively. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in catalytic activity and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the intestine of infected cats evolve over the course of infection, and the largest difference in the enriched pathways was observed at 96 hpi. The anti-T. gondii defense response of the feline host was mediated by Major Histocompatibility Complex class I, proteasomes, heat-shock proteins and fatty acid binding proteins. CONCLUSIONS: This study revealed novel host factors, which may be critical for the successful establishment of an intracellular niche during T. gondii infection in the definitive feline host.

First report on the occurance of intestinal Entamoeba spp. In pigs In China.

Three Entamoeba spp. including E. suis, zoonotic E. polecki, and E. histolytica, have been described in pigs to date. However, little is known about the molecular epidemiology of these neglected parasites in pigs globally. In this study we surveyed the occurrence and molecular epidemiology of porcine Entamoeba spp. in pigs in eastern China and evaluated their zoonotic potential. Five hundred fresh fecal samples, collected from seven pig farms in Anhui province, eastern China,were examined for the presence of E. histolytica, E. suis, and E. polecki ST1 and ST3 infections by a combination of nested PCR targeting the small subunit ribosomal DNA gene and subsequent sequencing.The overall occurrence of Entamoeba spp. was 45.8% (229/500). Infection with E. polecki ST1 (38.2%; 191/500) was the most common, followed by E. polecki ST3 (10.0%; 50/500), and E. suis (0.8%; 4/500). No E. histolytica infection was detected. Double infections with E. polecki ST1 and E. suis, and with E. polecki ST1 and ST3 were found in two (0.4%) and 14 (2.8%) samples, respectively. No age predisposition to infection with Entamoeba spp. was observed. PCR and subsequent sequencing confirmed the validity and feasibility of the nested PCR method used in this study in identifying species/subtypes of porcine Entamoeba spp.This is the first report to describe the occurrence and molecular epidemiology of Entamoeba species in pigs in China. The presence of two zoonotic E. polecki subtypes implies that pigs can be reservoirs for human E. polecki infections. More studiess are needed to better understand the transmission and public health significance of porcine Entamoeba spp.

Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs.

Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species.

Molecular epidemiological surveillance to assess emergence and re-emergence of tick-borne infections in tick samples from China evaluated by nested PCRs.

An investigation was performed to detect eight pathogens in ticks collected from grass tips or animals in the southern, central and northeast regions of China. DNA samples extracted from ticks were collected from ten different locations in eight provinces of China and subjected to screening for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Ehrlichia spp., Rickettsia spp., Babesia/Theileria spp., Ehrlichia ruminantium, Coxiella burnetii, and Francisella tularensis, using nested PCR assays and sequencing analysis. The results indicated that Borrelia spp., Rickettsia spp., and Babesia/Theileria spp. were detected in all of the investigated provinces. Ehrlichia spp. was also found in all of the surveyed areas, except Guangxi, Luobei and Tonghe counties in Heilongjiang province. The average prevalence of these pathogens was 18.4% (95% CI=12.8-42.5), 60.3% (95% CI=18.2-65.3), 26.0% (95% CI=25.8-65.1), and 28.7% (95% CI=5.6-35.2), respectively. A sequencing analysis of the pCS20 gene of E. ruminantium revealed an E. ruminantium-like organism (1/849, 0.1%, 95% CI=0-0.3) in one tick DNA sample extracted from Rhipicephalus (Boophilus) microplus in Hunan. In addition, Borrelia americana in Ixodes persulcatus, Babesia occultans in Haemaphysalis qinghaiensis and both Rhipicephalus sanguineus and an Ehrlichia muris-like organism in R. (B.) microplus was detected, possibly for the first time in China. Four DNA sequences closely related to Borrelia carolinensis and/or Borrelia bissettii from Haemaphysalis longicornis, Candidatus Rickettsia principis from H. qinghaiensis, and I. persulcatus and Ehrlichia canis (named E. canis-like) from Haemaphysalis bispinosa were also detected in this work.

Morphological Characteristics of Normal and Gynandromorphic Hyalomma asiaticum Schulze and Schlottke, 1930.

Gynandromorphic ticks are extremely rare, and often attract parasitologists' attention. During our examination of tick specimens, an engorged gynandromorph of Hyalomma asiaticum was noticed. This is the first record of gynandromorphic ticks from China. In this study, several important morphological structures of normal and gynandromorphic H. asiaticum were analyzed. Comparing to the normal H. asiaticum, the gynandromorphic specimen was a typical bipartite protogynander. Its right side showed normal female characteristics, whereas the left side had normal male traits. Different from other gynandromorphic ticks containing 1 anus, this tick reported here had 2 complete anuses, and the anus of the male part had a single adanal plate.

Identification and characterization of microRNAs by deep-sequencing in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks.

Hyalomma anatolicum anatolicum (H.a. anatolicum) (Acari: Ixodidae) ticks are globally distributed ectoparasites with veterinary and medical importance. These ticks not only weaken animals by sucking their blood but also transmit different species of parasitic protozoans. Multiple factors influence these parasitic infections including miRNAs, which are non-coding, small regulatory RNA molecules essential for the complex life cycle of parasites. To identify and characterize miRNAs in H.a. anatolicum, we developed an integrative approach combining deep sequencing, bioinformatics and real-time PCR analysis. Here we report the use of this approach to identify miRNA expression, family distribution, and nucleotide characteristics, and discovered novel miRNAs in H.a. anatolicum. The result showed that miR-1-3p, miR-275-3p, and miR-92a were expressed abundantly. There was a strong bias on miRNA, family members, and nucleotide compositions at certain positions in H.a. anatolicum miRNA. Uracil was the dominant nucleotide, particularly at positions 1, 6, 16, and 18, which were located approximately at the beginning, middle, and end of conserved miRNAs. Analysis of the conserved miRNAs indicated that miRNAs in H.a. anatolicum were concentrated along three diverse phylogenetic branches of bilaterians, insects and coelomates. Two possible roles for the use of miRNA in H.a. anatolicum could be presumed based on its parasitic life cycle: to maintain a large category of miRNA families of different animals, and/or to preserve stringent conserved seed regions with active changes in other places of miRNAs mainly in the middle and the end regions. These might help the parasite to undergo its complex life style in different hosts and adapt more readily to the host changes. The present study represents the first large scale characterization of H.a. anatolicum miRNAs, which could further the understanding of the complex biology of this zoonotic parasite, as well as initiate miRNA studies in other related species such as Haemaphysalis longicornis and Rhipicephalus sanguineus of human and animal health significance.

Detection of Toxoplasma gondii oocysts in soils in northwestern China using a new semi-nested PCR assay.

BACKGROUND: Toxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China. RESULTS: The one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 10(2) T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0-1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%. CONCLUSIONS: The present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.

Molecular evidence for piroplasms in wild Reeves' muntjac (Muntiacus reevesi) in China.

DNA from liver samples of 17 free-ranging wild Reeves' muntjac (Muntiacus reevesi) was used for PCR amplification of piropalsm 18S rRNA gene. Of 17 samples, 14 (82.4%) showed a specific PCR product which were cloned and sequenced. BLAST analysis of the sequences obtained showed similarities to Babesia sp., Theileria capreoli, Theileria uilenbergi and Theileria sp. BO302-SE. Phylogenetic analysis showed that the Babesia sp. detected in the present study was distantly separated from known Babesia species of wild and domestic animals. Six sequences showed 100% similarity to T. capreoli while five sequences were separated from all known Theileria species and constituted an independent clade with Theileria sp. BO302-SE derived from roe deer in Italy; two sequences were close to T. uilenbergi with 97% similarity. This is the first description of hemoparasite infection in free-ranging wild Reeves' muntjac in China. Our results indicate that wild Reeves' muntjac may play an important reservoir role for hemoparasites.

Preliminary Analysis of Taenia multiceps Metacestode Antigens by Two-Dimensional Electrophoresis.

BACKGROUND: Taenia multiceps is a cestode parasite with its larval stage (metacestode), Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestock causing cerebralis coenurosis. Since, treatment of coenurosis with chemotherapy showed little effect and surgical removal of cysts is not advisable in field conditions, vaccination is useful to control coenurosis. Previous study indicated that immunization with T. multiceps metacestode antigens could induce protection in sheep against coenurosis, so the aim of this study was to identify T. multiceps metacestode antigens in order to find potential vaccine development candidates for further study. METHODS: The protein extracts from the larval T. multiceps were analyzed by two-dimensional electrophoresis (2-DE) and characterized by mass spectrometry. RESULTS: A total of 150 protein spots were detected with isoelectric point (pI) value from 4.97 to 9.65 and molecular weight from 14 to 98 kDa. Twenty-two protein identities were determined by mass spectrometry and 15 unique proteins were obtained. Functional annotation revealed that some of these proteins are involved in catalytic activity, binding, metabolic, cellular process and stress response. Among these molecules are antioxidant proteins (peroxiredoxin and glutathione-S-transferase), glycolytic enzymes (malate dehydrogenase and enolase), proteins with chaperone activity (heat shock protein 70 and small heat shock protein), and structural proteins (actin, actin modulator protein and paramyosin). CONCLUSION: The identification of T. multiceps metacestode protein will provide valuable information to elucidate their specific roles in the parasitism and screen new targets for vaccine development.

RPS8--a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. We compared the intron-exon structure and DNA sequences of the RPS8 gene from Babesia and Theileria spp. isolates in China. Similar to 18S rDNA, the 40S ribosomal protein S8 gene, RPS8, including both coding and non-coding regions is a useful and novel genetic marker for defining species boundaries and for inferring phylogenies because it tends to have little intra-specific variation but considerable inter-specific difference. However, more samples are needed to verify the usefulness of the RPS8 (coding and non-coding regions) gene as a marker for the phylogenetic position and detection of most Babesia and Theileria species, particularly for some closely related species.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Molecular characterization of Taenia multiceps isolates from Gansu Province, China by sequencing of mitochondrial cytochrome C oxidase subunit 1.

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.

Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

Sequence analysis of Theileria annulata surface protein in Chinese isolates.

OBJECTIVE: To study the TaSP polymorphism in three Chinese isolates of Theileria annulata. METHODS: The isolates from Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region were cultured in RPMI 1640 medium. TaSP gene was amplified from genomic DNA extracted from schizonts using polymerase chain reaction (PCR) and sequenced. Its amino acid sequence comparison was carried out with Clustal W2 multiple sequence alignment program. Molecular component and motif prediction were performed with online servers. RESULTS: The comparison of TaSP amino acid sequences of the three isolates showed that the central region (aa position 38-161) predicted to be the highly immunogenetic domain was polymorphic both in size and amino acid sequence, while the N-terminal (first 37 aa) and C-terminal (last 154 aa) parts were strongly conserved. Phylogenetic analysis and percentage identity revealed that the Chinese isolates were closely related to the isolates from Turkey, but quite different from those of India, Morocco and Tunisia. More importantly, variability was noticed among Chinese isolates, which caused both the location and number's differences of motif (casein kinase II phosphorylation sites) among three TaSP sequences. CONCLUSION: TaSP polymorphism exists in the Chinese isolates of T. annulata.