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Abstract Hybrid mapping is a powerful approach to efficiently identify and characterize genes regulated through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we perform a comprehensive multi-omic characterization of regulatory variation across the brain, liver, muscle, and placenta through four developmental stages. We produce one of the largest multi-omic datasets in pigs to date, including 16 whole genome sequenced individuals, as well as 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We develop a read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We show that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identify 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. We integrate methylation, chromatin accessibility, and gene expression data to show that allele specific expression can be explained in great part by allele specific methylation and/or chromatin accessibility. This study provides a comprehensive characterization of regulatory variation across multiple tissues and developmental stages in pigs.

ASFV C315R is homologous to the transcription factor TFIIB of large unclassified DNA viruses, and H359L is identical to the subunit 3 (RPB3) of eukaryotic RNA polymerase II. The C315R and H359L may play an important role in ASFV replication and transcription. Here, we evaluated the biological function of the C315R and H359L genes during virus replication in vitro and during infection in pigs. Results showed that C315R and H359L are highly conserved among ASFV genotype II strains; quantitative PCR (qPCR) and western blotting analyses revealed that C315R and H359L are early transcribed genes prior to viral DNA replication, but their protein expression is delayed. The immunofluorescence and western blotting analysis revealed that both proteins localized in the cell cytoplasm and nucleus at 24 h post infection, however, pH359L was mainly detected in the cell cytoplasm. Furthermore, overexpression of pH359L in MA104 cells significantly increased viral titer, RNA transcription levels, and viral protein expression levels, while overexpression of pC315R slightly enhanced ASFV replication. In contrast, siRNA targeting ASFV-H359L or C315R reduced replication efficiency in porcine macrophage culture compared to the parent ASFV-CN/SC/2019, demonstrating that C315R and H359L genes are necessary for ASFV replication. Finally, the functional role of C315R or H359L on PKR and eIF2α phosphorylation status and SG formation, as well as cytokine production were evaluated. These studies demonstrated that C315R and H359L are involved in virus replication processes in swine and play important roles in ASFV replication.

Abstract 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.

Abstract Background The establishment of a robust gut microbiota in piglets during their early developmental stage holds the potential for long-term advantageous effects. However, the optimal timeframe for introducing probiotics to achieve this outcome remains uncertain. Results In the context of this investigation, we conducted a longitudinal assessment of the fecal microbiota of 63 piglets at three distinct pre-weaning time points. Simultaneously, we gathered vaginal and fecal samples from 23 sows. Employing 16S rRNA gene and metagenomic sequencing methodologies, we conducted a comprehensive analysis of the fluctuation patterns in microbial composition, functional capacity, interaction networks, and colonization resistance within the gut microbiota of piglets. As the piglets progressed in age, discernible modifications in intestinal microbial diversity, composition, and function were observed. A source-tracking analysis unveiled the pivotal role of fecal and vaginal microbiota derived from sows in populating the gut microbiota of neonatal piglets. By D21, the microbial interaction network displayed a more concise and efficient configuration, accompanied by enhanced colonization resistance relative to the other two time points. Moreover, we identified three strains of Ruminococcus sp. at D10 as potential candidates for improving piglets' weight gain during the weaning phase. Conclusions The findings of this study propose that D10 represents the most opportune juncture for the introduction of external probiotic interventions during the early stages of piglet development. This investigation augments our comprehension of the microbiota dynamics in early-life of piglets and offers valuable insights for guiding forthcoming probiotic interventions.

Objective Loin muscle area (LMA) is an important target trait of pig breeding. This study aimed to identify single nucleotide polymorphisms (SNPs) and genes associated with LMA in the Duroc×(Landrace×Yorkshire) crossbred pigs (DLY). Methods A genome-wide association study was performed using the Illumina 50K chip to map the genetic marker and genes associated with LMA in 511 DLY pigs (255 boars and 256 sows). Results After quality control, we detected 35,426 SNPs, including six SNPs significantly associated with LMA in pigs, with MARC0094338 and ASGA0072817 being the two key SNPs responsible for 1.77% and 2.48% of the phenotypic variance of LMA, respectively. Based on previous research, we determined two candidate genes (growth hormone receptor [GHR] and 3-oxoacid Co A-transferase 1 [OXCT1]) that are associated with fat deposition and muscle growth and found further additional genes (MYOCD, ARHGAP44, ELAC2, MAP2K4, FBXO4, FBLL1, RARS1, SLIT3, and RANK3) that are presumed to have an effect on LMA. Conclusion This study contributes to the identification of the mutation that underlies quantitative trait loci associated with LMA and to future pig breeding programs based on marker-assisted selection. Further studies are needed to elucidate the role of the identified candidate genes in the physiological processes involved in LMA regulation.

Marbofloxacin (MBF) was once widely used as a veterinary drug to control diseases in animals. MBF residues in animal food endanger human health. In the present study, an immunochromatographic strip assay (ICSA) utilizing a competitive principle was developed to rapidly detect MBF in beef samples. The 50% inhibitory concentration (IC50) and the limit of detection (LOD) of the ICSAs were 2.5 ng/mL and 0.5 ng/mL, respectively. The cross-reactivity (CR) of the MBF ICSAs to Ofloxacin (OFL), enrofloxacin (ENR), norfloxacin (NOR), and Ciprofloxacin (CIP) were 60.98%, 32.05%, 22.94%, and 23.58%, respectively. The CR for difloxacin (DIF) and sarafloxacin (SAR) was less than 0.1%. The recovery rates of MBF in spiked beef samples ranged from 82.0% to 90.4%. The intra-assay and interassay coefficients of variation (CVs) were below 10%. In addition, when the same authentic beef samples were detected in a side-by-side comparison between the ICSAs and HPLC‒MS, no statistically significant difference was observed. Therefore, the proposed ICSAs can be a useful tool for monitoring MBF residues in beef samples in a qualitative and quantitative manner.

Abstract Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valuable in modern pork production. However, genetic improvement has been slow due to high phenotyping costs. In this study, whole genome sequence (WGS) data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction (GBLUP) for meat quality in large-scale crossbred commercial pigs. Results We produced WGS data (18,695,907 SNPs and 2,106,902 INDELs exceed quality control) from 1,469 sequenced Duroc × (Landrace × Yorkshire) pigs and developed a reference panel for meat quality including meat color score, marbling score, L* (lightness), a* (redness), and b* (yellowness) of genomic prediction. The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population. Using different marker density panels derived from WGS data, accuracy differed substantially among meat quality traits, varied from 0.08 to 0.47. Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39% to 75%. We optimized the marker density and found medium- and high-density marker panels are beneficial for the estimation of heritability for meat quality. Moreover, we conducted genotype imputation from 50K chip to WGS level in the same population and found average concordance rate to exceed 95% and r 2 = 0.81. Conclusions Overall, estimation of heritability for meat quality traits can benefit from the use of WGS data. This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

The bovine IgG1 Fc receptor (boFcγRIII) is a homologue to human FcγRIII (CD16) that binds bovine IgGI with medium–low affinity. In order to identify the Fc-binding site on the bovine IgG1 Fc receptor (boFcγRIII), peptides derived from the second extracellular domain (EC2) of boFcγRIII were synthesized and conjugated with the carrier protein. With a Dot-blot assay, the ability of the peptides to bind bovine IgG1 was determined, and the IgG1-binding peptide was also identified via truncation and mutation. The minimal peptide AQRVVN corresponding to the sequence 98–103 of boFcγRIII bound bovine IgG1 in Dot-blot, suggesting that it represents a linear ligand-binding site located in the putative A–B loop of the boFcγRIII EC2 domain. Mutation analysis of the peptide showed that the residues of Ala98, Gln99, Val101, Val102 and Asn103 within the Fc-binding site are critical for IgG1 binding on boFcγRIII. The functional peptide identified in this paper is of great value to the IgG–Fc interaction study and FcR-targeting drug development.

Abstract 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.

Abstract Background Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers. Methods We evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites. Results Transfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months. Conclusions In our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj. Graphical abstract

Abstract This study aimed to construct a widely accepted prognostic nomogram in Chinese high-grade osteosarcoma (HOS) patients aged ≤ 30 years to provide insight into predicting 5-year overall survival (OS). Data from 503 consecutive HOS patients at our centre between 12/2012 and 05/2019 were retrospectively collected. Eighty-four clinical features and routine laboratory haematological and biochemical testing indicators of each patient at the time of diagnosis were collected. A prognostic nomogram model for predicting OS was constructed based on the Cox proportional hazards model. The performance was assessed by the concordance index (C-index), receiver operating characteristic curve and calibration curve. The utility was evaluated by decision curve analysis. The 5-year OS was 52.1% and 2.6% for the nonmetastatic and metastatic patients, respectively. The nomogram included nine important variables based on a multivariate analysis: tumour stage, surgical type, metastasis, preoperative neoadjuvant chemotherapy cycle, postoperative metastasis time, mean corpuscular volume, tumour-specific growth factor, gamma-glutamyl transferase and creatinine. The calibration curve showed that the nomogram was able to predict 5-year OS accurately. The C-index of the nomogram for OS prediction was 0.795 (range, 0.703–0.887). Moreover, the decision curve analysis curve also demonstrated the clinical benefit of this model. The nomogram provides an individualized risk estimate of the 5-year OS in patients with HOS aged ≤ 30 years in a Chinese population-based cohort.

African swine fever virus (ASFV) is a large and very complex DNA virus. The major capsid protein p72 is the most predominant structural protein and constitutes the outmost icosahedral capsid of the virion. In the present study, the nanobodies against ASFV p72 protein were screened from a camelid immune VHH library by phage display technique. Nine distinct nanobodies were identified according to the amino acid sequences of the complementary determining regions (CDRs), and contain typical amino acid substitutions in the framework region 2 (FR2). Six nanobodies were successfully expressed in E. coli, and their specificity and affinity to p72 protein were further evaluated. The results showed that nanobodies Nb25 had the best affinity to both recombinant and native p72 protein of ASFV. The Nb25 possesses an extremely long CDR3 with 23 amino acids compared with other nanobodies, which may allow this nanobody to access the hidden epitopes of target antigen. Furthermore, the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV in a sandwich immunoassay, and its application as a biosensor to target virus in PAM cells was verified by an immunofluorescence assay. Nanobodies have been proven to possess many favorable properties with small size, high affinity and specificity, easier to produce, low costs and deep tissue penetration that make them suitable for various biotechnological applications. These findings suggest that nanobody Nb25 identified herein could be a valuable alternative tool and has potential applications in diagnostic and basic research on ASFV.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal infectious disease of pigs and causes great socioeconomic losses globally. The reliable diagnostic method is critical for prevention and control of the disease. In this study, an improved Luciferase immunosorbent assay (LISA) for detecting ASF was developed using the cell lysates containing ASFV p35 protein fused with a reporter Nano-luciferase (p35-Luc protein). The improved method avoids the complicate procedures of immobilizing the serum samples with protein G in the normal LISA method, and replaced by directly coating the serum samples with carbonate buffer, therefore reduces the productive cost and simplifies the operation procedures. The p35-Luc LISA exhibited high specificity for anti-ASFV sera while no cross-reactions with the sera against other swine viruses. The detection limit of the p35-Luc LISA was shown to be at least four times higher than that of the p35 based indirect ELISA established in our lab. The receiver operating characteristic (ROC) analysis showed the 96.36% relative specificity and 96.97% relative sensitivity of the p35-Luc LISA with the cutoff values of 3.55 as compared to the commercial Ingezim p72-ELISA kit. Furthermore, a total of 248 serum samples were tested by both the p35-Luc LISA and commercial Ingezim p72-ELISA kit, and there was a high degree of agreement (97.6%, kappa = 0.9753) in the performance of the two assays. Collectively, the improved LISA based on the p35-Luc protein could be used as a rapid, ultrasensitive, cost-effective and reliable diagnostic tool for serological survey of ASF in pig farms.

In recent years, African swine fever (ASF) has caused a devastating blow to the swine industry globally. Since no effective vaccine is available, strict biosafety measures and rapid diagnosis are the most effective strategies for ASF control. ASFV p30 is one of the most antigenic viral proteins that have been widely used in the field for serological diagnosis of ASF infection. In this study, we developed a luciferase immunoprecipitation system (LIPS) assay for the detection of ASFV antibodies in pig serum using Gaussia luciferase (GLuc)-tagged p30 as a diagnostic antigen. The optimal GLuc-p30 input of 107 luminance units (LU) and optimal serum dilution factor of 1/100 were set to achieve the highest P/N ratio. Based on 87 ASFV-positive and negative pig sera, the cutoff value of the S/N ratio could be set between 2.298 and 30.59 to achieve 100% sensitivity and 100% specificity. Moreover, the diagnostic sensitivity of this LIPS is comparable to that of a commercial enzyme-linked immunosorbent assay (ELISA) and the specificity of LIPS is even superior to the tested ELISA. In conclusion, we have established a LIPS assay for ASFV antibody detection, which could be a potential method for ASFV diagnosis in laboratories and farms.

ABSTRACT African swine fever (ASF), an acute, severe, highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and boars, has a mortality rate of up to 100%. Because effective vaccines and treatments for ASF are lacking, effective control of the spread of ASF remains a great challenge for the pig industry. Host epigenetic regulation is essential for the viral gene transcription. Bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic “readers” critical for gene transcription regulation. Among these proteins, BRD4 recognizes acetylated histones via its two bromodomains (BD1 and BD2) and recruits transcription factors, thereby playing a pivotal role in transcriptional regulation and chromatin remodeling during viral infection. However, how BET/BRD4 regulates ASFV replication and gene transcription is unknown. Here, we randomly selected 12 representative BET family inhibitors and compared their effects on ASFV infection in pig primary alveolar macrophages (PAMs). These were found to inhibit viral infection by interfering viral replication. The four most effective inhibitors (ARV-825, ZL0580, I-BET-762, and PLX51107) were selected for further antiviral activity analysis. These BET/BRD4 inhibitors dose dependently decreased the ASFV titer, viral RNA transcription, and protein production in PAMs. Collectively, we report novel function of BET/BRD4 inhibitors in inducing suppression of ASFV infection, providing insights into the role of BET/BRD4 in the epigenetic regulation of ASFV and potential new strategies for ASF prevention and control. IMPORTANCE Due to the continuing spread of the ASFV in the world and the lack of commercial vaccines, the development of improved control strategies, including antiviral drugs, is urgently needed. BRD4 is an important epigenetic factor and has been commonly used for drug development for tumor treatment. Furthermore, the latest research showed that BET/BRD4 inhibition could suppress replication of virus. In this study, we first showed the inhibitory effect of agents targeting BET/BRD4 on ASFV infection with no significant host cytotoxicity. Then, we found four BET/BRD4 inhibitors that can inhibit ASFV replication, RNA transcription, and protein synthesis. Our findings support the hypothesis that BET/BRD4 can be considered as attractive host targets in antiviral drug discovery against ASFV.

Abstract 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.

We report a new competitive lateral flow immunoassay (LFIA) for hapten detection, which uses antibodies to bind antigens from a sample and form a coloured immune complex due to artificial antigens conjugated with gold nanoparticles; these were intercepted with SPA. Here, 2,4-dichlorophenoxyacetic acid (2,4-D) and gentamicin (GM) were selected as model analytes. The visual limit of detection of the 2,4-D strip was 25 ng/mL in tap water; the GM strip was 3.13 ng/mL in milk. This approach exhibits more than a 40-fold lower detection limit for 2,4-D and 3.2-fold lower for GM, which is compared to traditional LFIA using the same reagents. This approach can accurately control the amount of antibody, protect the activity of antibody and enhance the detection signal, thus improving the sensitivity of the LFIA and providing more and more sensitive methods for the development of LFIAs. Highlights This study developed a new method of LFIA that could improve the sensitivity of LFIA without adding any reagent. The antibodies are unlabelled; thus, the immunological properties of the antibodies are not destroyed by antibody folding during the labelling process. Sufficient gold-labelled antigens ensure that each antibody bound to the test line can combine two gold nanoparticles, thereby amplifying the overall signal.

Abstract Background Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. Methods The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. Results A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. Conclusions To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.

A rapid immunochromatographic lateral flow strip test (ILFST) of competitive format for the detection of diclofenac (DCF) in medicinal wine samples was developed. The half-inhibitory concentration (IC50) values of the ILFST for DCF and the limit of detection (LOD) in DCF-spiked samples were 0.66 and 0.15 ng/mL, respectively. Recoveries of DCF from spiked medicinal wine samples were from 84.4% to 85.9% within an assay (intra-assay) and from 83.7% to 88.4% between assays (inter-assay). The coefficients of variation (CV) for intra-assay and inter-assay were 4.10–7.56% and 4.71–8.94%, respectively. These results indicated that the ILFST was highly sensitive and re-producible. The same authentic medicinal wine samples were tested by both ILFST and high-performance liquid chromatography (HPLC) respectively. The results showed no significant differences between the two methods. Therefore, the ILFST can be widely used as a convenient tool for both qualitative and quantitative detection of DCF residues in medicinal wine samples.

Aquaporins (AQPs) are important functional proteins and are widely present in the cell membrane of almost all organisms, mediating transmembrane transport of liquid and other solutes. Much is known about the molecular characterization of AQPs in other tick species; however, nothing is known about them in Haemaphysalis qinghaiensis. In this study, we first sequenced the transcript variants of AQPs in H. qinghaiensis (HqAQPs), analyzed the biological structure features of AQPs, and investigated the pattern of gene expression of the AQP gene of H. qinghaiensis in different tick tissues and stages to predict their biological functions. In conclusion, four AQP transcript variants (i.e., HqAQP1-1, HqAQP1-2, HqAQP1-3, and HqAQP1-4) of H. qinghaiensis were found, and the sequences were comparable with its orthologs from the reported tick species. Gene expression of AQPs in different tick tissues and stages showed the higher expression level in salivary glands and gut of adult female, as well as in the female and nymph than in Malpighian tubules, ovary, male, larvae, and egg. Further studies will be performed to evaluate the function of HqAQPs against H. qinghaiensis infestation on animals.

African swine fever virus (ASFV) infection can result in lethal disease in pigs. ASFV encodes 150-167 proteins, of which only approximately 50 encoded viral structure proteins are functionally known. ASFV also encodes some nonstructural proteins that are involved in the regulation of viral transcription, viral replication and evasion from host defense. However, the understanding of the molecular correlates of the severity of these infections is still limited. The purpose of this study was to compare host and viral gene expression differences and perform functional analysis in acutely infected, dead and cohabiting asymptomatic pigs infected with ASFV by using RNA-Seq technique; healthy pigs were used as controls. A total of 3,760 and 2,874 upregulated genes and 4,176 and 2,899 downregulated genes were found in healthy pigs vs. acutely infected, dead pigs or asymptomatic pigs, respectively. Additionally, 941 upregulated genes and 956 downregulated genes were identified in asymptomatic vs. acutely infected, dead pigs. Different alternative splicing (AS) events were also analyzed, as were gene chromosome locations, and protein-protein interaction (PPI) network prediction analysis was performed for significantly differentially expressed genes (DEGs). In addition, 30 DEGs were validated by RT-qPCR, and the results were consistent with the RNA-Seq results. We further analyzed the interaction between ASFV and its host at the molecular level and predicted the mechanisms responsible for asymptomatic pigs based on the selected DEGs. Interestingly, we found that some viral genes in cohabiting asymptomatic pigs might integrate into host genes (DP96R, I73R and L83L) or remain in the tissues of cohabiting asymptomatic pigs. In conclusion, the data obtained in the present study provide new evidence for further elucidating ASFV-host interactions and the ASFV infection mechanism and will facilitate the implementation of integrated strategies for controlling ASF spread.

Wildlife is involved in the maintenance and transmission of various tick-borne pathogens. The objective of the present study was to determine the occurrence and diversity of tick-borne pathogens in free-ranging wild animals collected from Tangjiahe National Nature Reserve of China. Blood or liver samples from 13 wild animals (5 takin, 3 Himalayan goral, 3 Reeves' muntjac, 1 forest musk deer, and 1 wild boar) were collected and screened for piroplasm, Anaplasma spp., Ehrlichia spp., and spotted fever group (SFG) rickettsiae by PCR-based on different gene loci. Three Theileria species, a potential novel Theileria parasite (Theileria sp. T4) and two Anaplasma species were identified in those wildlife. Theileria capreoli was found in Himalayan goral, Reeves' muntjac, and forest musk deer; Theileria luwenshuni, Theileria uilenbergi, and a potential novel, Theileria parasite (Theileria sp. T4), were identified in takin. Meanwhile, Anaplasma bovis was identified in Himalayan goral, takin, Reeves' muntjac, forest musk deer, and wild boar; Anaplasma phagocytophilum and related strains was found in takin, Reeves' muntjac, and forest musk deer. All wildlife included in this study was negative for Babesia, Anaplasma ovis, Anaplasma marginale, Ehrlichia, and SFG rickettsiae. Moreover, coinfection involving Theileria spp. and Anaplasma spp. was observed in eight wild animals. This study provided the first evidence of tick-borne pathogens in free-ranging wild animals from the nature reserve, where contact between domestic and wild animals rarely occurs.

Abstract Background Small ruminants are important hosts for various tick species and tick-associated organisms, many of which are zoonotic. The aim of the present study was to determine the presence of tick-borne protozoans and bacteria of public health and veterinary significance in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. Methods The occurrence of piroplasms, Anaplasma phagocytophilum, A. bovis, A. marginale, A. capra, A. ovis, Ehrlichia spp. and spotted fever group rickettsiae was molecularly investigated and analyzed in 134 goats and 9 free ranging C. pygargus living in close proximity. Results Piroplasm DNA was detected in 16 (11.9%) goats and 5 C. pygargus. Sequence analysis of 18S rRNA sequences identified 3 Theileria species (T. luwenshuni, T. capreoli and T. cervi). Four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) were identified in goats and C. pygargus. Anaplasma ovis and A. bovis were detected in 11 (8.2%) and 6 (4.5%) goats, respectively; A. phagocytophilum, A. bovis and A. capra were found in 3, 7 and 3 C. pygargus, respectively. Sequence analysis of 16S rRNA sequences revealed the presence of 5 different genetic variants of A. bovis in goats and C. pygargus, while the analysis of 16S rRNA and gltA sequence data showed that A. capra isolates identified from C. pygargus were closely related to the genotype identified from sheep and Haemaphysalis qinghaiensis, but differed with the genotype from humans. Anaplasma/Theileria mixed infection was observed in 2 (1.5%) goats and 5 C. pygargus, and co-existence involving potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. All samples were negative for A. marginale, Ehrlichia spp. and SFG rickettsiae. Conclusions These findings report the tick-borne pathogens in goats and C. pygargus, and a greater diversity of these pathogens were observed in wild animals. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The contact between wild and domestic animals may increase the potential risk of spread and transmission of tick-borne diseases.

Abstract 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.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

Study Design: Meta-analysis. Background: Although some new insights have been offered for clinical and scientific relevance, minor progress has been made in osteosarcoma treatment after a dramatic survival improvement in the late 1980s with the addition of chemotherapy to surgery. Intensified chemotherapy strategies have been suggested to increase the survival rate of patients with osteosarcoma. We performed this study to access whether intensified chemotherapy strategiesincreased survival outcomes of osteosarcoma patients compared with conventional chemotherapy strategies. Methods: MEDLINE/PubMed, EMBASE, BIOSIS Previews, and Cochrane Library were searched from database set up to October2016. Randomized controlled trials (RCTs) and comparative clinical trials (CCTs) on intensified versus conventional chemotherapy strategies for osteosarcoma patients met the inclusion criteria, and the methodological quality standard were retrieved and reviewed. Data on participant characteristics, interventions, follow-up period, and outcomes were extracted from the included studies and analyzed by Review Manager 5.3. Results: 12 studies (8 RCTs and 4CCT) involving 4112 patients were selected. There were no significant differences between intensified and conventional chemotherapy strategies group in 3-year event-free survival (OR, 1.01; 95% CI, [0.74–1.37]; P = 0.97), 5-year event-free survival (OR, 1.00; 95% CI, [0.86–1.17]; P = 0.97), and 5-year overall survival (OR, 1.04; 95% CI, [0.87–1.26]; P = 0.64), and good histologic response to preoperative chemotherapy (OR, 1.12; 95% CI, [0.78–1.60]; P = 0.55). Pooled analysis of local recurrence rate showed that local recurrence rate was significantly decreased in the intensified group compared with that in the conventional group (OR, 0.60; 95% CI, [0.42–0.85]; P = 0.004). Conclusions: Intensified chemotherapy might not be a preferred treatment for all of the osteosarcoma patients. Keywords: Meta-analysis, High dosage, Chemotherapy, Osteosarcoma

Osteosarcoma is the most common primary malignant bone tumor; its standard treatment includes neoadjuvant chemotherapy combined with surgery. Neoadjuvant chemotherapy has significantly improved the 5-year survival and limb salvage rates in osteosarcoma since the 1870s. The survival rate of patients with limb salvage was not inferior to that of amputees, and therefore, limb salvage has become the main surgical option for patients with osteosarcoma. The 5-year survival rate for osteosarcoma has plateaued. However, new advances in limb salvage therapy in osteosarcoma, including adjuvant chemotherapy, ablation techniques, bone transport techniques, and computer navigation techniques, are now available. This report summarizes the recent advances in limb salvage therapy for osteosarcoma over the past decade.

Abstract Background Anaplasma marginale is an important tick-transmitted rickettsial pathogen of cattle, with worldwide distribution and an important economic impact. The genetic diversity of A. marginale strains has been extensively characterized in different geographical regions throughout the world, while information is limited on studies in China. This study was carried out to determine the prevalence and genetic diversity of A. marginale strains in cattle from ten provinces of China. Methods A total of 557 blood samples from cattle were collected and screened for the occurrence of A. marginale by PCR based on the msp4 gene. The partial msp1a gene containing tandem repeat sequences was further amplified from msp4 positive samples. The Msp1a amino acid repeats were identified, and genetic variation of A. marginale strains was characterized based on the variation in the repeated portion of Msp1a. Results Our results showed that 31.6% of 557 cattle were positive for A. marginale. The infection rates of A. marginale varied considerably from 0 to 96.9% in different sampling regions. Sequence analysis revealed that two msp4 sequence variants of A. marginale exist in cattle. One hundred and three msp1a sequences were obtained and permitted to identify 42 Msp1a tandem repeats, 21 of which were not previously published for A. marginale. Moreover, 61 A. marginale genotypes were identified based on the structure of Msp1a tandem repeats. Conclusions Anaplasma marginale is widely distributed in China and a high prevalence of infection was observed in cattle. The geographical strains of A. marginale were molecularly characterized based on the structure of Msp1a tandem repeats. Forty-two Msp1a tandem repeats and 61 genotypes of A. marginale were identified. This study, for the first time, revealed the genetic diversity of A. marginale strains in cattle in China.

Abstract Background Anaplasma ovis is one of the tick-transmitted pathogens of small ruminants. It causes ovine anaplasmosis and widely distributed in the world. In contrast to extensive worldwide genetic diversity of A. marginale and A. phagocytophilum, there are few reports on the classification of A. ovis strains. This study was conducted to investigate the occurrence and characterize A. ovis strains from goats and sheep from 12 provinces in China. Methods The occurrence of A. ovis DNA was tested in 552 goats and sheep, by PCR based on the msp4 gene. Positive samples were used for the amplification of the msp1a gene of A. ovis. The Msp1a amino acid repeats were further identified and used for the characterization of A. ovis strains. Results The results showed that 79 (14.3%) goats and sheep were positive for A. ovis. The infection rates of A. ovis among different study sites ranged from 0 to 100%, and were significantly higher in sheep (26.6%, 45/169) than in goats (8.9%, 34/383) (χ 2 = 21.403, df = 1, P

Abstract Background Ovine anaplasmosis is a tick-borne disease that is caused by Anaplasma ovis in sheep and goats. The pathogen is widely distributed in tropical and subtropical regions of the world. At present, diagnosis of the disease mainly depends on microscopy or nucleic acid based molecular tests, although a few serological tests have been applied for the detection of A. ovis infection. Results Here we describe the identification of an A. ovis protein that is homologous to the A. marginale appendage-associated protein (AAAP). We expressed a recombinant fragment of this protein for the development of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of A. ovis. Anaplasma ovis-positive serum showed specific reactivity to recombinantly expressed AAAP (rAAAP), which was further confirmed by the rAAAP ELISA, which also demonstrated no cross-reactivity with sera from animals infected with A. bovis or other related pathogens in sheep and goats. Testing antibody kinetics of five experimentally infected sheep for 1 year demonstrated that the rAAAP ELISA is suitable for the detection of early and persistent infection of A. ovis infections. Investigation of 3138 field-collected serum samples from 54 regions in 23 provinces in China demonstrated that the seroprevalence varied from 9.4% to 65.3%, which is in agreement with previous reports of A. ovis infection. Conclusions An A. ovis derived antigenic protein, AAAP, was identified and the antigenicity of the recombinant AAAP was confirmed. Using rAAAP an indirect ELISA assay was established, and the assay has been proven to be an alternative serological diagnostic tool for investigating the prevalence of ovine anaplasmosis of sheep and goats.

Abstract Background Tick-borne diseases currently represent an important issue for global health. A number of emerging tick-transmitted microbes continue to be discovered, and some of these are already identified as the cause of human infections. Over the past two decades, Anaplasma phagocytophilum is considered to be mainly responsible for human anaplasmosis. However, a novel zoonotic pathogen provisionally named “Anaplasma capra” has recently been identified in China. In this study, we did an active surveillance of A. capra in goats and sheep in different geographical regions of China. Methods The presence of A. capra was determined by nested PCR in 547 blood samples collected from goats and sheep from 24 counties distributed in 12 provinces in China. The molecular characterization of A. capra isolates in sheep and goats was achieved based on four conventional genetic markers (16S rRNA, gltA, groEL and msp4 genes). Results Anaplasma capra was identified in 75 of 547 animals, with an overall prevalence of 13.7%. The infection rates in the survey sites ranged from 0 to 78.6%, and were significantly different (P

Abstract Background Bovine theileriosis is a common disease transmitted by ticks, and can cause loss of beef and dairy cattle worldwide. Here, an indirect enzyme-linked immunosorbent assay (iELISA) based on Theileria luwenshuni surface protein (TlSP) was developed and used to carry out a seroepidemiological survey of bovine theileriosis in northern China. Methods We used the BugBuster Ni-NTA His•Bind Purification Kit to purify recombinant TlSP (rTlSP), which was subsequently analyzed by Western Blotting to evaluate cross-reactivity with other pathogen-positive sera. The iELISA method based on rTlSP was successfully developed. Sera from 2005 blood samples were tested with the rTlSP-iELISA method, and blood smears from these samples were observed by microscopy. Results The specificity of iELISA was 98.9%, the sensitivity was 98.5%, and the cut-off was selected as 24.6%. Western Blot analysis of rTlSP confirmed that there were cross-reactions with Theileria luwenshuni, Theileria uilenbergi, Theileria ovis, Theileria annulata, Theileria orientalis and Theileria sinensis. The epidemiological survey showed that the highest positive rate of bovine theileriosis was 98.3%, the lowest rate was 84.1%, and the average positive rate was 95.4% by iELISA. With microscopy, the highest positive rate was 38.9%, the lowest rate was 5.1%, and the relative average positive rate was 13.7%. Conclusions An rTlSP-iELISA was developed to detect circulating antibodies against bovine Theileria in northern China. This is the first report on the seroprevalence of bovine theileriosis in northern China, and it also provides seroepidemiological data on bovine theileriosis in China.

Abstract Background Anaplasma spp. are tick-transmitted bacteria that infect a wide variety of wild and domestic animals. These pathogens exhibit a high degree of biological diversity, broad geographical distribution, and represent a serious threat to veterinary and public health worldwide. Results A novel Anaplasma species was identified in Haemaphysalis qinghaiensis (Ixodidae) in northwestern China and was molecularly characterized by comparison of 16S rRNA, gltA, and groEL gene sequences. Of the 414 samples tested, 24 (5.8%) were positive for this Anaplasma species. On the basis of the 16S rRNA gene, this organism has been found to be closely related to and exhibit the highest sequence similarity with A. capra (99.8–99.9%) that was identified in goats and humans in northern China, but was distinct from other known Anaplasma species. Sequence analysis of the gltA and groEL genes revealed that this Anaplasma species was distinct from A. capra considering the lower sequence identity (88.6–88.7% for gltA and 90.6–91.0% for groEL) and a divergent phylogenetic position. Therefore, we described this Anaplasma species as A. capra-like bacteria. Conclusions The present study reports a potential novel Anaplasma species closely related to A. capra in H. qinghaiensis in northwestern China. The zoonotic potential of A. capra-like bacteria needs to be further determined.

Abstract Background Anaplasma phagocytophilum is wide spread throughout the world and impacts both human and animal health. Several distinct ecological clusters and ecotypes of the agent have been established on the basis of various genetic loci. However, information on the genetic variability of A. phagocytophilum isolates in China represents a gap in knowledge. The objective of this study was to determine the prevalence and genetic characterization of A. phagocytophilum in small ruminants in central and southeastern China. Methods The presence of A. phagocytophilum was determined in 421 blood samples collected from small ruminants by PCR. Positive samples were genetically characterized based on 16S rRNA and groEL genes. Statistical analyses were conducted to identify ecotypes of A. phagocytophilum strains, to assess their host range and zoonotic potential. Results Out of 421 sampled small ruminants, 106 (25.2%) were positive for A. phagocytophilum. The positive rate was higher in sheep (35.1%, 40/114) than in goats (26.4%, 66/307) (P

Abstract Background Theileria uilenbergi and T. luwenshuni are tick-borne protozoan parasites, transmitted by Haemaphysalis qinghaiensis and H. longicornis, respectively. They are the main causative agents of theileriosis in small ruminants in China. The disease has resulted in severe economic losses and hindered the development of sheep and goat husbandry industry in the endemic regions. Methods In this study, a colloidal gold-based immunochromatographic strip (ICS) was developed for the detection of T. uilenbergi and/or T. luwenshuni infections. A recombinant T. uilenbergi immunodominant protein (rTuIP) was used as antigen for the ICS. The nitrocellulose membrane was incubated with rTuIP on the test (T) line and anti-rTuIP antiserum on the control (C) line, respectively. The rTuIP conjugated to colloidal gold particles was used as the detection system for visualization of the lines. Then the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were assembled onto a backing plate in the appropriate order. Results The ICS was able to detect antibodies in the sera of animals experimentally infected with T. uilenbergi from 14 to 85 days. It also reacted with the serum from T. luwenshuni infected sheep. However, there was no cross-reactivity with sera from animals infected with Babesia motasi and Anaplasma ovis. Comparison of the ICS with the rTuIP antigen based indirect enzyme-linked immunosorbent assays (ELISA) using test field samples showed good correlations with 93.1 % (81/87) sensitivity and 100 % (40/40) specificity, respectively, with an almost perfect agreement (Kappa = 0.895, P

The apicomplexan parasite Babesia bovis is a tick-borne intracellular hemoprotozoan parasite that is widespread across China. Genetic diversity is an important strategy used by parasites to escape the immune responses of their hosts. In our present study, 575 blood samples, collected from cattle in 10 provinces, were initially screened using a nested PCR (polymerase chain reaction) for detection of B. bovis infection. To perform genetic diversity analyses, positive samples were further amplified to obtain sequences of three B. bovis merozoite surface antigen genes (MSA-1, MSA-2b, MSA-2c). The results of the nested PCR approach showed that an average of 8.9% (51/575) of cattle were positive for B. bovis infection. Phylogenetic analyses of the predicted amino acid sequences revealed that unique antigen variants were formed only by Chinese isolates. Our findings provide vital information for understanding the genetic diversity of B. bovis in China.

A lateral flow immunochromatographic strip test was developed for rapid and sensitive on-site detection of hexoestrol (HES) residues in fish samples with colloidal gold labelling of the anti-HES monoclonal antibody. The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC50) of the strip in the detection of fish extract samples was confirmed to be 1.86 µg kg−1, and the limit of detection value was 0.62 µg kg−1. For intra-assay and inter-assay reproducibility, recoveries of HES-spiked samples ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91–4.64% and 4.24–5.17%, respectively. High-performance liquid chromatography was employed to confirm the performance of the strip. The strip test takes less than 10 min, and thus provides a repaid method for on-site detection of HES residues.

Abstract Background Babesiosis is an important haemoparasitic disease, caused by the infection and subsequent intra-erythrocytic multiplication of protozoa of the genus Babesia that impacts the livestock industry and animal health. The distribution, epidemiology and genetic characterization of B. bigemina, B. bovis, and B. ovata in cattle in China as well as the prevalence of these protozoan agents were assessed. Methods A total of 646 blood specimens from cattle, dairy cattle and yaks from 14 provinces were collected and tested for the presence of the three Babesia species via a specific nested PCR assay based on the rap-1 and ama-1 genes. The PCR results were confirmed by DNA sequencing. Gene sequences and the genetic characterization were determined for selected positive samples from each sampling area. Results Of a total of 646 samples, 134 (20.7 %), 60 (9.3 %) and 10 (1.5 %) were positive for B. bovis, B. bigemina and B. ovata infections, respectively. Mixed infections were found in 7 of 14 provinces; 43 (6.7 %) samples were infected with B. bovis and B. bigemina. Three samples (0.5 %) exhibited a co-infection with B. bovis and B. ovata, and 6 (0.9 %) were infected with all three parasites. The rap-1a gene of B. bovis indicated a high degree of sequence heterogeneity compared with other published rap-1a sequences worldwide and was 85–100 % identical to B. bovis rap-1a sequences in Chinese isolates. B. bigemina rap-1c and B. ovata ama-1 genes were nearly identical, with 97.8–99.3 % and 97.8–99.6 % sequence identity, respectively, in GenBank. Conclusions Positive rates of B. bovis and B. bigemina infection are somewhat high in China. The B. bovis infection in yaks was first reported. The significant sequence heterogeneity in different variants of the rap-1a gene from Chinese B. bovis isolates might be a great threat to the cattle industry if RAP-1a protein is used as immunological antigen against Babesia infections in China. The data obtained in this study can be used to plan effective control strategies against babesiosis in China.

Abstract Background Bovine babesiosis and theileriosis is an important hemoprotozoal disease in cattles and yaks in tropical and subtropical regions leading to significant economic losses. In the field, the risk of co-infection between the bovine Babesia and Theileria species is very high. Thus, it is necessary to develop a simple, accurate, rapid and cost-effective method for large-scale epidemic investigation, in particular for the detection of co-infection in field. Methods In this study, DNA sequences of a ribosomal protein S8 (RPS8) gene from eight species of cattle piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The eight Theileria and Babesia species could be differentiated by digesting the RPS8 PCR product with Mbo I. Results The sensitivity of the PCR assays was 0.1 pg DNA for Babesia species but 1 pg DNA for Theileria species. The clearly different size of the PCR-RFLP products allowed for a direct discrimination between eight bovine Theileria and Babesia species (T. annulata, T. sinensis, T. sergenti, B. ovata, B. bovis, B. bigemina, B. major and Babesia species Kashi isolate). Conclusion Our results indicated that the established method based on the RPS8 gene was a reliable molecular diagnostic tool for the simultaneous detection and identification of bovine Babesia and Theileria species in China, which could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.

Abstract Background To date, anaplasmosis has been reported to be a subclinical disease in Indian and Arabian one-humped camels (Camelus dromedarius) and llamas (Lama glama). However, no information on Anaplasma infection in two-humped Bactrian camels (Camelus bactrianus) in China has been published to date. The aim of this study was to investigate the prevalence of Anaplasma spp. in domestic Bactrian camels and ticks in Xinjiang, China. Findings A total of 382 ticks were collected from the Bactrian camels and from environmental sources. Of these, 84 were morphologically identified as belonging to the Rhipicephalus sanguineus group and genetically identified (12S rDNA, 16S rDNA and the cytochrome c oxidase 1 genes) as R. sanguineus group ticks (temporally designated as Rhipicephalus sp. Xinjiang). PCR testing showed that 7.2 % (20/279) of the camels harbored Anaplasma platys DNA. However, microscopic examination revealed no A. platys inclusions in blood smears from the camels. The PCR prevalence of A. platys DNA was 9.5 % (6/63) in Rhipicephalus sp. Xinjiang from the Bactrian camels and 14.3 % (3/21) in Rhipicephalus sp. Xinjiang from the vegetation. A. platys DNA was not detected by PCR in other tick species (Hyalomma asiaticum, Dermacentor niveus and Hyalomma dromedarii), and no other Anaplasma species were detected in these samples. Conclusions This is the first report of A. platys in Bactrian camels in Xinjiang, China. The moderate positivity observed indicates that these animals might be a natural host for this pathogen in China.

[i]Borrelia burgdorferi[/i] sensu lato, the etiological agent of Lyme disease, is tick transmitted and has a wide range of mammalian reservoirs in nature, including both wild and domestic animals. To understand the seroprevalence of [i]B. burgdorferi[/i] s.l. in small ruminants will add value to the risk analysis of Lyme disease. The current study was intended to map the potential endemic regions of Lyme disease by large-scale investigation of sera from sheep and goats. In this study, a total of 2,758 serum samples from sheep and goats in 21 provinces located in 40 different districts of China were tested for antibodies against [i]B. burgdorferi[/i] s.l. by enzyme-linked immunosorbent assay. The results of this survey indicated that the overall prevalence of [i]B. burgdorferi[/i] s.l. infection ranges from 5.3 to 63.5 % (mean: 26.3%), and the infection was found in all provinces investigated. Generally, the positive rate declined from the south (34.7% in south and 32.4% in southwest) towards the north of China (18.4% in north, 16.5% in northeast and 17.2% in northwest). A significant difference was also observed in the infection rate between south and north (33.2% versus 17.4%, [i]P[/i]

Abstract Background Anaplasmosis is caused by obligate intracellular bacteria in the genus Anaplasma. These bacterial pathogens are transmitted by ticks and impact both human and animal health. This study was conducted to determine the prevalence and molecular characterization of Anaplasma spp. in ruminants sampled in Xinjiang, northwest China. Methods A survey was performed in August 2012 in rural areas of six counties in Xinjiang province. A total of 250 blood samples from ruminants were collected and tested for the presence of Anaplasma spp. by PCR. Positive samples were genetically characterized based on the 16S rRNA and msp4 genes. Results The results showed a high prevalence of Anaplasma spp. in ruminants, with at least three different Anaplasma species detected (A. phagocytophilum, A. bovis and A. ovis). The mean prevalence of single infection with each species was 17.6% (A. phagocytophilum), 4.8% (A. bovis) and 40.5% (A. ovis). Coinfection occurred in 20 (8.0%) animals. Phylogenetic analysis of the 16S rRNA gene of A. bovis and A. phagocytophilum revealed a higher degree of genetic diversity for the latter. The results for A. ovis showed genotypic variation among geographic regions in China. In addition, a closely related isolate to the canine pathogen A. platys was identified in ruminants. Conclusions This survey revealed a high prevalence of Anaplasma sp. infections in sheep and cattle in the northwestern border regions of China, indicating the potential risk of transboundary disease.

Abstract Background Theileria and Anaplasma are especially important emerging tick-borne pathogens of animals and humans. Molecular surveys and identification of the infectious agents in Mongolian gazelle, Procapra gutturosa are not only crucial for the species’ preservation, but also provide valuable information on parasite and bacterial epidemiology. Findings A molecular surveillance study was undertaken to assess the prevalence of Theileria spp. and Anaplasma spp. in P. gutturosa by PCR in China. Theileria luwenshuni, A. bovis, A. phagocytophilum, and A. ovis were frequently found in P. gutturosa in China, at a prevalence of 97.8%, 78.3%, 65.2%, and 52.2%, respectively. The prevalence of each pathogens in the tick Haemaphysalis longicornis was 80.0%, 66.7%, 76.7%, and 0%, respectively, and in the tick Dermacentor niveus was 88.2%, 35.3%, 88.2%, and 58.5%, respectively. No other Theileria or Anaplasma species was found in these samples. Rickettsia raoultii was detected for the first time in P. gutturosa in China. Conclusions Our results extend our understanding of the epidemiology of theileriosis and anaplasmosis in P. gutturosa, and will facilitate the implementation of measures to control these tick-borne diseases in China.

Abstract Background Wildlife is an important natural reservoir of many tick-borne pathogens. These agents have an impact on the health of humans and other animals throughout the world. This study was conducted to determine whether and what species of tick-borne agents had infected wild birds collected from Guangxi, in southwest China. Findings Liver samples obtained from wild birds were tested for the presence of tick-borne pathogens by PCR assays and sequencing of the flagellin and 16S rRNA genes. Borrelia garinii was detected in Eurasian collared doves (2/57, 3.5 %) from among the 95 wild birds. Anaplasma phagocytophilum was detected in Eurasian collared doves (2/57, 3.5 %) and Eurasian eagle owls (2/13, 15.4 %). Ehrlichia chaffeensis and a potential novel Anaplasma sp. were identified in Common pheasant (1/12, 8.3 %). These results suggest the involvement of birds in the cycle of tick-borne diseases. To our knowledge, this is the first study to document infection of birds with B. garinii, A. phagocytophilum, E. chaffeensis and the novel Anaplasma sp. in China. Conclusions Tick-borne zoonotic bacteria B. garinii, A. phagocytophilum and E. chaffeensis, and a potential novel Anaplasma sp., were identified in wild birds in southwest China. The presence of these agents in birds increases the potential spread over long distances and the risk of transmission of infection from birds to new hosts, including humans.

Babesiosis, the hemolytic disease caused by Babesia, which is a tick-transmitted obligate intraerythrocytic protozoan parasite. This disease is responsible for significant mortality and morbidity rates and enormous economic losses to the livestock industry in tropical and subtropical regions worldwide. In this study, blood samples were collected from 141 pet dogs from Gansu, China, and analyzed for Babesia or Theileria spp. infection using specific PCR and sequencing based on 18S rRNA gene fragments. The results indicated that 18S rRNA gene sequences from 11 samples were similar to the 18S rRNA gene sequences in Babesia canis vogeli (2) and Theileria sinensis (9). The total infected rates of B. canis vogeli and T. sinensis were 1.4% (2/141) and 6.4% (9/141), respectively. This represents the first molecular report of T. sinensis in dogs worldwide and of B. canis vogeli in dogs from Gansu province of China. Furthermore, the finding of T. sinensis in dogs may represent the common infection of this parasite occurring in Gansu.

Abstract Background Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. Methods The primers used in the LAMP assay were based on the mitochondrial nad 5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People’s Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. Results The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. Conclusion The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Abstract Background Theileria and Babesia protozoan parasites are transmitted mainly by tick vectors. These parasites cause heavy economic losses to the live-stock industry, as well as affecting the health of wild animals in parasite-endemic areas. Identification of infectious agents in wild animals is not only crucial for species preservation, but also provides valuable information on parasite epidemiology. Here, we conducted a molecular surveillance study in Northwestern China to assess the prevalence of blood pathogens in cervids. Methods PCR analysis and microscopic evaluation of blood smears to detect Theileria- and Babesia-related diseases in Cervidae were conducted, in which 22 blood samples from red deer (n = 22) in Qilian Mountain and 20 from sika deer (n = 20) in Long Mountain were collected and tested for the presence of Theileria and Babesia. The 18S rRNA gene was amplified, and selected polymerase chain reaction (PCR)-positive samples were sequenced for species identification. Results PCR revealed that 9.1% of the Qilian Mountain samples and 20% of the Long Mountain samples were positive for Theileria uilenbergi; 90.09% of the Qilian Mountain samples (n = 22) were positive for T. capreoli, but all of the Long Mountain samples (n = 20) were negative for T. capreoli; no other Theileria or Babesia species were found. PCR showed that T. uilenbergi and T. capreoli were present in red deer in Qilian Mountain, while only T. uilenbergi was found in sika Deer in Long Mountain. The 18S rRNA gene sequences were aligned against the corresponding GenBank sequences of known isolates of Theileria and Babesia and subjected to phylogenetic analysis. The phylogenetic tree showed that the newly isolated Theileria spp. could be classified as belonging to two clades: one group belonged to the same clade as T. uilenbergi, the other to a clade containing T. capreoli. Conclusions Our results provide important data to increase understanding of the epidemiology of Cervidae theileriosis, and will assist with the implementation of measures to control theileriosis transmission to Cervidae and small ruminants in central China.

Obligate intracellular bacteria belonging to the genus Anaplasma spp. are responsible for causing a hemolytic disease called anaplasmosis in animals, as well as in humans. This study was aimed at the molecular identification and genetic analysis of responsible causative agents of anaplasmosis beyond those already reported. A survey was performed during July and August 2018 in the Jhang District, Punjab, Pakistan. Four hundred and fifty blood samples from asymptomatic, tick-infested cattle were collected on FTA cards and tested for the Anaplasma spp. presence using nested-polymerase chain reaction (PCR) methods. The 16S ribosomal RNA gene sequences generated from the positive samples were used for genetic analysis of Anaplasma spp. The nested-PCR results showed the presence of two Anaplasma spp. with an overall prevalence rate of 10.44%, where the prevalence of A. bovis and A. phagocytophilum was 7.78% and 2.66%, respectively. The study portrayed new molecular data on the prevalence of Anaplasma spp. in the studied cattle population, indicating a potential threat to the human population as well.