OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81 kcal/mol, while the docking energy for meropenem was - 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28 kcal/mol, compared with - 4.60 kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Identification and Characterization of Nanobodies from a Phage Display Library and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most lethal and devastating diseases of domestic and wild swine. The continual spread and frequent outbreaks of ASF have seriously threatened the pig and pig-related industries, causing great socioeconomic losses at unprecedented proportions. Although ASF has been documented for a century, no effective vaccine or antiviral treatment is currently available. Nanobodies (Nbs) derived from heavy-chain-only antibodies in camelids have been discovered to be effective as therapeutics and robust biosensors in imaging and diagnostic applications. In the present study, a high-quality phage display library containing specific Nbs raised against ASFV proteins was successfully constructed, and 19 nanobodies specific to ASFV p30 were preliminarily identified by phage display technology. After extensive evaluation, nanobodies Nb17 and Nb30 were employed as immunosensors and applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ASFV in clinical specimens. This immunoassay showed a detection limit of approximately 1.1 ng/mL target protein and 102.5 hemadsorption (HAD50/mL) of ASFV and exhibited high specificity with no cross-reaction with the other porcine viruses tested. The performances of the newly developed assay and a commercial kit in testing 282 clinical swine samples were very similar (93.62% agreement). However, the novel sandwich Nb-ELISA showed higher sensitivity than the commercial kit when serial dilutions of ASFV-positive samples were tested. The present study describes a valuable alternative technique for the detection and surveillance of ASF in endemic regions. Furthermore, additional nanobodies specific to ASFV may be developed using the generated VHH library and employed in different biotechnology fields.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Multiple-probe-assisted DNA capture and amplification for high-throughput African swine fever virus detection.

African swine fever (ASF) is one of the most devastating infectious diseases affecting domestic pigs and wild boar. The grave socio-economic impact of African swine fever infection at a global level makes large-scale rapid and robust diagnosis a critical step towards effective control. Here, we describe multiple-probe-assisted DNA capture and amplification technology (MADCAT) - a novel, sensitive, simple, and high-throughput method for detecting ASFV directly from whole blood or other complex matrices. Through a unique DNA capture approach which specifically captures the target DNA onto 96-well plate for subsequent amplification, MADCAT abandons the complicated extraction protocol and achieves ultrafast and high-throughput detection. The sample-to-result time for 96 samples is about 90 min, as compared with the 3-4 h time of the conventional real-time qPCR method. The limit of detection (LOD) of MADCAT is 0.5 copies/µL blood and is 5 times more sensitive than an extraction-based qPCR assay when testing serially diluted whole blood samples. The assay is 100% specific against other common swine pathogens. In the clinical diagnosis of 96 field samples, all 22 positive samples were correctly identified with lower Ct values than extraction-based qPCR, confirming its high diagnostic sensitivity (100%). Owing to its high-throughput, specific high sensitivity, and direct detection features, MADCAT shows great potential for use in large-scale ASFV surveillance and monitoring for effective disease control. KEY POINTS: • No nucleic acid extraction, 100% capture efficiency, and high-throughput • Ultra-high sensitivity of 0.5 DNA copies/µL or 6 DNA copies/reaction • The sample-to-answer time for 96 samples is about 90 min.

Comparative genomic analysis of Babesia duncani responsible for human babesiosis.

BACKGROUND: Human babesiosis, caused by parasites of the genus Babesia, is an emerging and re-emerging tick-borne disease that is mainly transmitted by tick bites and infected blood transfusion. Babesia duncani has caused majority of human babesiosis in Canada; however, limited data are available to correlate its genomic information and biological features. RESULTS: We generated a B. duncani reference genome using Oxford Nanopore Technology (ONT) and Illumina sequencing technology and uncovered its biological features and phylogenetic relationship with other Apicomplexa parasites. Phylogenetic analyses revealed that B. duncani form a clade distinct from B. microti, Babesia spp. infective to bovine and ovine species, and Theileria spp. infective to bovines. We identified the largest species-specific gene family that could be applied as diagnostic markers for this pathogen. In addition, two gene families show signals of significant expansion and several genes that present signatures of positive selection in B. duncani, suggesting their possible roles in the capability of this parasite to infect humans or tick vectors. CONCLUSIONS: Using ONT sequencing and Illumina sequencing technologies, we provide the first B. duncani reference genome and confirm that B. duncani forms a phylogenetically distinct clade from other Piroplasm parasites. Comparative genomic analyses show that two gene families are significantly expanded in B. duncani and may play important roles in host cell invasion and virulence of B. duncani. Our study provides basic information for further exploring B. duncani features, such as host-parasite and tick-parasite interactions.

Development of a p72 trimer-based colloidal gold strip for detection of antibodies against African swine fever virus.

African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV. KEY POINTS : • ASFV p72 trimers were successfully generated. • A colloidal gold strip was developed based on ASFV p72 trimers. • The strip is appropriate for detecting ASFV antibodies in the field.

The first molecular identification and phylogenetic analysis of tick-borne pathogens in captive wild animals from Lohi Bher zoo, Pakistan.

Tick-borne pathogens are causing severe diseases in livestock, wild animals, and humans. Wild animals play a crucial role in tick-borne pathogens' transmission life cycle by serving as reservoir hosts or intermediate hosts, posing a continuous risk for domestic animals and humans. The presence of tick-borne pathogens is often ignored in wild animals kept in zoos, which is a public health concern. In the present study, we investigated these pathogens in tick-infested captive wild animals at the Lohi Bher zoo, Pakistan. Blood samples were collected from 22 animals, which include urials (4) (Ovis aries vignei), blackbucks (3) (Antilope cervicapra), fallow deer (1) (Dama dama), hog deer (6) (Axis porcinus), chinkaras (4) (Gazella bennettii), white tiger (2) (Panthera tigris tigris), a giraffe (Giraffa camelopardalis), and African lions (2) (Panthera leo). The samples were screened for Piroplasm and Anaplasma spp. by polymerase chain reaction targeting different gene loci. We detected three Theileria spp. and one Anaplasma sp. from the investigated captive wild animals. The Theileria sp. dama gazelle was detected from chinkara, Theileria sp. NG-2012b from chinkara and giraffe and T. parva from African lion, and Anaplasma bovis was identified in a giraffe. Moreover, Theileria sp. and Anaplasma sp. coinfection was detected in one giraffe. Overall, this study shows that Theileria spp. and Anaplasma spp. are circulating in captive wild animals, which can play an important role in their spread. Further studies are required to monitor tick-borne pathogens in zoo animals and their potential to spread from exotic wild captive animals to local wild and domestic.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses.

BACKGROUND: H7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. METHODS: The H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared. RESULTS: The analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples. CONCLUSION: The McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Establishment of a transient transfection system for Babesia sp. Xinjiang using homologous promoters.

Babesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (BspXJ), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remains unclear. Firstly, a plasmid bearing the elongation factor-1 alpha promoter and the firefly luciferase reporter gene and rap stop region were transfected into BspXJ by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program settings were evaluated to confirm the best for BspXJ transient transfection, and a series of different amounts of plasmid DNA were transfected to generate relatively high luminescence values. Finally, the activities of four promoters derived from BspXJ were evaluated using the developed transient transfection system. After evaluating of various transfection parameters, the human T cell nucleofector solution, program V-024 and 20 µg of plasmid DNA were selected as the most favorable conditions for BspXJ transient transfection. These findings provide critical information for BspXJ genetic manipulation, an essential tool to identify virulence factors and to further elucidate the basic biology of this parasite.

A strip test for the optical determination of influenza virus H3 subtype using gold nanoparticle coated polystyrene latex microspheres.

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10 nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. Graphical abstract .

A rapid colloidal gold-based immunochromatographic strip assay for monitoring nitroxynil in milk.

BACKGROUND: Nitroxynil (NIT) is a veterinary drug against hepatic fluke disease for food-producing cattle and sheep. NIT has a long half-life time in animals since it is highly bound to plasma protein. Therefore NIT possibly remains in animal edible tissues or milk due to drug abuse. In this study, a specific murine monoclonal antibody (mAb) against NIT was prepared and an immunochromatographic strip assay based on the mAb was developed for screening NIT in milk. RESULTS: The affinity constant of the anti-NIT mAb was 2.93 × 1010 and the anti-NIT mAb had almost no cross-reactivity with other analogs, so that it showed good specificity. The cutoff value of this test strip was considered to be 50 ng mL-1 by the naked eye. When detected by the strip reader, the half maximum inhibition concentration (IC50 ) of the immunoassay strip was calculated to be 5.716 ng mL-1 and the limit of detection (LOD) was 1.146 ng mL-1 . Intra-assay recoveries from 88.80 to 97.13% were obtained, with the highest coefficient of variation (CV) at 9.01%; inter-assay recoveries ranged from 84.60 to 106.87%, with the highest CV at 9.93%. CONCLUSION: The operative procedure of the proposed method can be completed within 10 min. The strip developed in this study was a practical tool for rapid semiquantitative and quantitative detection of NIT in milk. This study suggested great potential for analytically monitoring NIT in other food samples. © 2019 Society of Chemical Industry.

The Anaplasma ovis genome reveals a high proportion of pseudogenes.

BACKGROUND: The genus Anaplasma is made up of organisms characterized by small genomes that are undergoing reductive evolution. Anaplasma ovis, one of the seven recognized species in this genus, is an understudied pathogen of sheep and other ruminants. This tick-borne agent is thought to induce only mild clinical disease; however, small deficits may add to larger economic impacts due to the wide geographic distribution of this pathogen. RESULTS: In this report we present the first complete genome sequence for A. ovis and compare the genome features with other closely related species. The 1,214,674 bp A. ovis genome encodes 933 protein coding sequences, the split operon arrangement for ribosomal RNA genes, and more pseudogenes than previously recognized for other Anaplasma species. The metabolic potential is similar to other Anaplasma species. Anaplasma ovis has a small repertoire of surface proteins and transporters. Several novel genes are identified. CONCLUSIONS: Analyses of these important features and significant gene families/genes with potential to be vaccine candidates are presented in a comparative context. The availability of this genome will significantly facilitate research for this pathogen.

Molecular detection and genetic diversity of Theileria orientalis in cattle in China.

The apicomplexan parasite Theileria orientalis is a tick-borne intracellular protozoan parasite that is widely distributed throughout China. It causes bovine theileriosis in infected cattle, which results in huge economic losses to the cattle industry. In this study, the infection status of T. orientalis was determined in 260 blood samples from cattle from seven provinces across China. Results of a major piroplasm surface protein (MPSP)-PCR assay revealed that an average of 36.5% (95/260) of cattle was positive for T. orientalis infection. Based on the MPSP gene sequences, phylogenetic analysis revealed that these isolates of T. orientalis comprised of eight MPSP types, 1, 2, 3, 4, 5, 7, N1, and N2. This is the first report of new T. orientalis MPSP genotypes N1 and N2 in cattle in China.

Molecular cloning, characterization and antigenicity of Babesia sp. BQ1 (Lintan) (Babesia cf. motasi) apical membrane antigen-1 (AMA-1).

Apical membrane antigen-1 (AMA-1) has been described as a potential vaccine candidate in apicomplexan parasites. Here we characterize the ama-1 gene. The full-length ama-1 gene of Babesia sp. BQ1 (Lintan) (BLTAMA-1) is 1785 bp, which contains an open reading frame (ORF) encoding a 65-kDa protein of 594 amino acid residues; by definition, the 5' UTR precedes the first methionine of the ORF. Phylogenetic analysis based on AMA-1 amino acid sequences clearly separated Piroplasmida from other Apicomplexa parasites. The Babesia sp. BQ1 (Lintan) AMA-1 sequence is most closely associated with that of B. ovata and B. bigemina, with high bootstrap value. A recombinant protein encoding a conserved region and containing ectodomains I and II of BLTAMA-1 was constructed. BLTrAMA-1-DI/DII proteins were tested for reactivity with sera from sheep infected by Babesia sp. BQ1 (Lintan). In Western-blot analysis, native Babesia sp. BQ1 (Lintan) AMA-1 proteins were recognized by antibodies raised in rabbits against BLTrAMA-1 in vitro. The results of this study are discussed in terms of gene characterization, taxonomy and antigenicity.

Molecular evidence of tick-borne pathogens in Hyalomma anatolicum ticks infesting cattle in Xinjiang Uygur Autonomous Region, Northwestern China.

Although tick-borne pathogens have been widely reported in ticks in China, there is little information available on the prevalence of information in Hyalomma ticks from cattle. This study aims to determine the occurrence of pathogens in Hyalomma anatolicum collected from cattle in Xinjiang Uygur Autonomous Region, China, by PCR, sequencing and phylogenetic analysis. Borrelia burgdorferi s.s., Rickettsia massiliae and Anaplasma bovis were identified, whereas DNA of Ehrlichia species and an Anaplasma platys-like pathogen were also detected. Our findings highlight the risk of infection of animals and humans with these pathogens in north-western China.

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay.

Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

First molecular survey and identification of Anaplasma spp. in white yaks (Bos grunniens) in China.

Anaplasmosis is caused by a group of obligate intracellular bacteria in the genus Anaplasma, which are transmitted by ticks and infect humans, domestic animals and wildlife. This study was conducted to determine the prevalence and molecular characterization of Anaplasma spp. in semi-wild white yaks sampled in Tianzhu Tibetan Autonomous County, northwest China. Out of 332 samples tested, 35 (10·9%) were positive for Anaplasma spp. The positive rates were 6·2% (20/322) and 5·3% (17/322) for Anaplasma bovis and Anaplasma phagocytophilum in white yaks, respectively. None of the sample was positive for Anaplasma marginale. Two (0·6%) samples were simultaneously positive to A. bovis and A. phagocytophilum. Sequence analysis of the 16S rRNA gene revealed two genotypes (ApG1 and ApG2) of A. phagocytophilum and two sequence types (ST1 and ST2) of A. bovis in white yaks. This study is the first to document the presence of Anaplasma in white yaks. Our findings extend the host range for Anaplasma species and provide more valuable information for the control and management of anaplasmosis in white yaks.

Evaluation of different nested PCRs for detection of Anaplasma phagocytophilum in ruminants and ticks.

BACKGROUND: Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in mammals, which has a broad geographical distribution and a high degree of clinical diversity. Currently, numerous PCR assays have been developed and used for the detection of A. phagocytophilum in various specimens. However, their performance varies. The aim of this study was to evaluate the performance of five nested PCR assays by detection of 363 ruminant and tick samples, and to select the most appropriate methods for the sensitive detection of A. phagocytophilum in environmental or clinical samples. RESULTS: Positive PCR results for A. phagocytophilum were obtained in 75 (20.7%), 42 (11.6%) and 19 (5.2%) specimens with primer sets EC (EC9/EC12a and SSAP2f/SSAP2r), EE (EE1/EE2 and EE3/EE4) and ge (ge3a/ge10r, ge9f/ge2), respectively. The amplification of template DNA with the primer set MSP (MAP4AP5/MSP4AP3, msp4f/msp4r) could not be obtained in both ruminants and ticks, and a low specificity of the EL primers [EL(569)F/EL(1193)R, EL(569)F/EL(1142)R] in tick samples was observed. Our results revealed that the nested PCR with primer set EC complementary to the 16S rRNA gene was the most sensitive assay for detection of A. phagocytophilum in ruminant and tick specimens. A. phagocytophilum was detected in 47 (35.1%) sheep, 12 (10.4%) cattle, and 17 (14.9%) ticks. Two A. phagocytophilum genotypes were identified, that varied between sheep and cattle in sample collection sites. CONCLUSIONS: This report provides more valuable information for the diagnosis and management of granulocytic anaplasmosis in China.

Comparative proteomic and bioinformatic analysis of Theileria luwenshuni and Theileria uilenbergi.

Theileria is an obligatory intraerythrocytic protozoan parasite that causes economic losses to the cattle, sheep and goats industry. However, very little information is available on the genomes, transcriptomes, and proteomes of the ovine parasites, Theileria luwenshuni and Theileria uilenbergi. Differences in protein expression between these species were investigated to better understand their biology. Parasites were digested with trypsin, and the resulting peptides labeled with isobaric tags for relative and absolute quantification, followed by LC-MS/MS. More than 670 proteins, classified into categories primarily related to cellular process (29.78%), metabolic process (28.80%), localization (5.22%) and biological regulation (5.00%), were identified. Seventy-one proteins were differentially expressed; T. luwenshuni had 39 proteins more highly expressed than in T. uilenbergi, whereas T. uilenbergi had 32 that were more highly expressed. Several proteins related to parasite virulence and invasion (cysteine proteinase, histone deacetylase, pyruvate kinase, small nuclear ribonucleoprotein and orotate phosphoribosyltransferase) were differentially expressed. Real-time quantitative PCR validated protein expression changes at the transcript level. This is the first report on protein expression for the two most economically important Theileria species in China, and our findings may provide novel opportunities for ovine and caprine theileriosis control.