Mapping a highly conserved linear neutralizing epitope at the N-terminus of the gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae subfamily, causes significant economic losses to the cattle industry worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an essential role in the viral entry into permissive cells and possibly cooperates with other envelope glycoproteins. The herpesvirus gD induces a protective immune response against diseases in cattle or animal models. Mapping epitopes on gD will facilitate the understanding of the BoHV-1 pathogenesis and development of alternative vaccines against various diseases associated with the virus. In this study, a monoclonal antibody (MAb), designated as 3C1, was generated using naive BoHV-1 in vaccination of mice, demonstrating that 3C1 was specific to gD and represents a neutralizing activity against BoHV-1 infection in Madin-Darby bovine kidney cells. Panels of overlapping gD recombinant proteins with glutathione S-transferase tag were prepared to define the epitope recognized by 3C1. The data demonstrated that the N-terminus of gD 23APRVTVYVD31 was recognized by 3C1. Furthermore, the 26VTVYVD31 motif was the minimal amino acid sequence for the recognition. The epitope identified in this study is highly conserved among the typical strains of BoHV-1 and BoHV-5, suggesting that this epitope may be useful in the diagnosis of diseases. In addition, the defined region on gD of BoHV-1 might be essential in viral entry upon comparison with the prototype virus in herpes simplex virus (Alphaherpesvirinae). The data will elucidate the roles of gD of BoHV-1 in viral entry and pathogenesis and its potential application for the development of vaccine candidates and diagnostic techniques based on the conserved epitopes on gD or in combination with those of other herpesvirus glycoproteins.

Protective immunity following vaccination with a recombinant multiple-epitope protein of bovine herpesvirus type I in a rabbit model.

Bovine herpesvirus type 1 (BoHV-1) causes considerable economic losses to the cow industry. Vaccination remains an effective strategy to control the diseases associated with BoHV-1. However, live vaccines present safety concerns, especially in pregnant cows; thus, nonreplicating vaccines have been developed to control the disease. The envelope glycoproteins of BoHV-1 induce a protective immune response. In this work, selected epitopes on glycoproteins gD, gC, and gB were constructed in triplicate with linker peptides. Vaccination of rabbits demonstrated that P2-gD/gC/gB with AAYAAY induced higher specific antibodies than that with GGGGS linker. P2-gD/gC/gB with AAYAAY linker was fused with bovine interleukin-6 (BoIL-6) or rabbit IL-6 (RaIL-6) and bacterially expressed. Rabbits were intramuscularly immunized with 100 µg of P2-gD/gC/gB-BoIL-6, P2-gD/gC/gB-RaIL-6, P2-gD/gC/gB, P2-gD/gC/gB plus BoIL-6, P2-(gD-a)3-BoIL-6, or P2-(gD-a)3 emulsified with ISA 206 adjuvant thrice at 3-week intervals. P2-gD/gC/gB-BoIL-6 generated a higher titer of BoHV-1-specific antibodies, neutralizing antibodies, interferon (IFN)-γ, and IL-4 compared with P2-gD/gC/gB plus BoIL-6, P2-gD/gC/gB-RaIL-6, or other formulation. P2-gD/gC/gB-BoIL-6 triggered similar levels of antibodies and significantly higher titer of IFN-γ and IL-4 compared with inactivated bovine viral diarrhea (BVD)-infectious bovine rhinotracheitis (IBR) vaccine. Rabbits vaccinated with P2-gD/gC/gB-BoIL-6 dramatically reduced viral shedding and tissue lesions in lungs and trachea after viral challenge and reactivation compared with those with P2-gD/gC/gB plus BoIL-6 or P2-gD/gC/gB-RaIL-6. P2-gD/gC/gB-BoIL-6 provided protective effects against viral shedding and tissue pathogenesis similar to those of the inactivated vaccine. The data confirmed the safety and immunogenicity of multiple-epitope recombinant protein and a potential vaccine candidate to control the disease, especially for pregnant cattle.

Preparation of porcine enterotoxigenic Escherichia coli (ETEC) ghosts and immunogenic analysis in a mouse model.

Enterotoxignenic Escherichia coli (ETEC)-associated colibacillosis causes high levels of morbidity and mortality in neonatal piglets. Vaccination is among effective strategy to fight against ETEC-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which substain subtle antigenic comformation in bacterial outer membrane. In this study, a BG vaccine was generated using porcine ETEC isolated strain DQ061 and evaluated its safety and immunogenicity in a mouse model. The recombinant bacteria were constructed by transformation of lysis plasmid pHH43 and generation of BGs was conducted in a lysis rate of 99.93% by incubation of the recombinant bacteria at 42 °C for 2 h. Mice were immunized subcutaneously twice in 2-week intervals with BGs, BGs emulsified with ISA 206 adjuvant, or formalin-inactivated ETEC vaccine after safety test. Mice with either of two BG vaccines developed higher titer of antibodies, secreted higher titer of interleukin 4, gamma interferon and alpha tumor necrosis factor after 2 doses than those with formalin-inactivated ETEC vaccine or those with adjuvant placebo (P < 0.01). The quantity of CD4+ and CD8+ T lymphocyte in spleen was higher in both BG groups than that in the inactivated vaccine group or adjuvant group 2 weeks post boost immunization (P < 0.05). The vaccinated mice were challenged intraperitoneally with 10 × LD50 dose of DQ061. Mice with the BGs plus adjuvant were completely protected against challenge, compared to 60% protection of mice with the inactivated vaccine. Mice exhibited decreased tissue lesion and reduced bacterial loads in the BGs groups by comparison with those with the inactivated vaccine or adjuvant only. Our results validated that the ETEC BGs bear high safety and immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model.

Generation of porcine Pasteurella multocida ghost vaccine and examination of its immunogenicity against virulent challenge in mice.

Pasteurella multocida (PM) causes a varity of clinical manifestation in domestic animals, even acute death. Vaccination is among effective strategy to prevent and control PM-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which sustain subtle antigenic comformation in bacterial outer-membrane and exhibit higher efficacy compared to inactivated vaccines. Here, a BG vaccine generated from the porcine PM reference strain CVCC446 (serotype B:2) was prepared upon lysis by E protein of bacteriophage PhiX174, and the safety and immunogenicity were evaluated its in a mouse model. Lysis rate was in 99.99% and the BG vaccine was completely inactivated by addition of freeze-dry procedure. Mice were immunized subcutaneously twice in 2-week intervals with BGs, or BGs plus adjuvant, or formalin-inactivated PM or an adjuvant control. Mice inoculated twice with BGs vaccines generated higher titer of antibodies, interleukin 4 and gamma interferon than those in the inactivated vaccine group or adjuvant placebo group (P < 0.05). CD4+ and CD8+ T lymphocyte levels in spleen were higher in both BG groups than inactivated vaccine group or adjuvant group. Mice administered with the BGs plus adjuvant were completely protected against intraperitoneal challenge with 10 × LD50 dose of virulent isolate and exhibited decreased tissue lesion and lower bacterial loads, which was superior to the inactivated vaccine. The results demonstrated safety of the BG vaccine and primary immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model and vaccine candidate.

Development and validation of a competitive ELISA based on bacterium-original virus-like particles of serotype O foot-and-mouth disease virus for detecting serum antibodies.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects all susceptible cloven-hoofed animals, resulting in considerable economic losses to animal industries worldwide. Numerous categories of enzyme-linked immunosorbent assays (ELISA) have been developed and widely used to evaluate herd immunity. Manufacturing inactivated FMD virus (FMDV) as a diagnostic antigen requires a facility with a high level of biosafety, but this requirement raises concern on viral leakage. In our previous study, bacterium-original FMD virus-like particles (VLPs) resemble the authentic FMDV and induce protective immunity against homologous viral challenges, thereby demonstrating that they are sufficiently safe without limitations on biosafety facilities and easily prepared. Herein, we developed a competitive ELISA (cELISA) based on FMDV-VLPs as a diagnostic antigen to evaluate herd immunity. The criterion of this cELISA was determined by detecting panels of positive sera with different antibody titers and negative sera. The working parameter of cELISA was optimized, and samples with a percentage inhibition of ≥ 50% were considered positive. The specificity of cELISA to test 277 serum samples with various antibody titers was 100%, and the sensitivity reached 96%. The coincidence rates of cELISA with a VDPro® FMDV and a PrioCHECK® FMDV type O antibody ELISA kit were 97.8% and 98.2%, respectively. Repeatability tests demonstrated that the coefficients of variation within and between runs were less than 7% and 14%, respectively. Our data demonstrated that cELISA based on bacterium-original VLPs had high specificity, sensitivity, and reproducibility. The cELISA could also be used for evaluating vaccination herd immunity effects, especially in developing countries.

Correction to: Brucellosis seroprevalence in ovine and caprine flocks in China during 2000-2018: a systematic review and meta-analysis.

The original article [1] contained a minor error whereby Table 1 was typeset incorrectly.

Brucellosis seroprevalence in ovine and caprine flocks in China during 2000-2018: a systematic review and meta-analysis.

BACKGROUND: Brucellosis remains one of the most common zoonotic diseases globally, with more than half million human cases reported annually. Brucellosis is an emerging and re-emerging disease in China since the 1990s. An infectious reservoir constituted by domestic animals with brucellosis, especially ovine and caprine herds, poses a significant threat to public health. The seroprevalence of brucellosis in sheep and goat flocks in a national context is unavailable so far. Therefore, we conducted this systematic review and meta-analysis to assess the overall status of brucellosis in sheep and goats in China in almost two decades. RESULTS: The pooled prevalence of brucellosis in ovine and caprine flocks in China increased in 2000-2009 (1.00%; 95% CI, 0.70-1.30) to 2010-2018 (3.20%; 95% CI, 2.70-3.60). The seroprevalence of brucellosis in sheep and goat flocks was higher in Eastern China, with 7.00% of positive rate, than that in any other region, especially Shandong province (18.70%). Brucellosis is highly endemic in some local regions. The high prevalence of brucellosis in agricultural regions is suggestive of a shift of geographic distribution. The pooled prevalence of brucellosis is higher in goat flocks than in sheep flocks in China. CONCLUSIONS: The overall data in this meta-analysis demands comprehensive intervention measures and further surveillance to facilitate the control of brucellosis in livestock. Further studies aimed at evaluating the risk factors associated with spreads of brucellosis in domestic animals unaddressed so far, and sufficient epidemiological data is important to the exploration and understanding of the prevalent status of brucellosis throughout the country and to disease control.

[Immunoreactivity and immunogenicity analysis of the recombinant cathepsin L-like protease of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunoreactivity of recombinant cathepsin L-like proteases (CatL) protein of Fasciola hepatica and its immunogenicity in SD rats. METHODS: The E. coli BL21(DE3) cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by methyl thiazolyl tetrazolium (MTT). RESULTS: The molecular weight of purified FhCatL was about Mr 42,000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization (1 : 102,400). The stimulation index of splenocytes in immunized group was 2.176 +/- 0.047, which was significantly higher than that of the control (1.171 +/- 0.032) (P<0.05). CONCLUSION: The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.

[Immunogenicity analysis of recombinant GST protein of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica (FhGST) in SD rats. METHODS: The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by MTT. RESULTS: The molecular weight of purified FhGST was about M(r) 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization (GMT 1:89 144). FhGST protein significantly enhanced the growth and proliferation of rat splenocytes. CONCLUSION: The recombinant FhGST protein induces specific immune response in SD rats.

Prevalence of Senecavirus A in pigs from 2014 to 2020: a global systematic review and meta-analysis.

BACKGROUND: Senecavirus A (SVA), a member of the family Picornaviridae, is newly discovered, which causes vesicular lesions, lameness in swine, and even death in neonatal piglets. SVA has rapidly spread worldwide in recent years, especially in Asia. OBJECTIVES: We conducted a global meta-analysis and systematic review to determine the status of SVA infection in pigs. METHODS: Through PubMed, VIP Chinese Journals Database, China National Knowledge Infrastructure, and Wanfang Data search data from 2014 to July 26, 2020, a total of 34 articles were included in this analysis based on our inclusion criteria. We estimated the pooled prevalence of SVA in pigs by the random effects model. A risk of bias assessment of the studies and subgroup analysis to explain heterogeneity was undertaken. RESULTS: We estimated the SVA prevalence to be 15.90% (1,564/9,839; 95% confidence interval [CI], 44.75-65.89) globally. The prevalence decreased to 11.06% (945/8,542; 95% CI, 28.25-50.64) after 2016. The highest SVA prevalence with the VP1-based RT-PCR and immunohistochemistry assay was 58.52% (594/1,015; 95% CI, 59.90-83.96) and 85.54% (71/83; 95% CI, 76.68-100.00), respectively. Besides, the SVA prevalence in piglet herds was the highest at 71.69% (119/166; 95% CI, 68.61-98.43) (p < 0.05). Moreover, our analysis confirmed that the subgroups, including country, sampling year, sampling position, detected gene, detection method, season, age, and climate, could be the heterogeneous factors associated with SVA prevalence. CONCLUSIONS: The results indicated that SVA widely exists in various countries currently. Therefore, more prevention and control policies should be proposed to enhance the management of pig farms and improve breeding conditions and the environment to reduce the spread of SVA.

Senecavirus A Enhances Its Adaptive Evolution via Synonymous Codon Bias Evolution.

Synonymous codon bias in the viral genome affects protein translation and gene expression, suggesting that the synonymous codon mutant plays an essential role in influencing virulence and evolution. However, how the recessive mutant form contributes to virus evolvability remains elusive. In this paper, we characterize how the Senecavirus A (SVA), a picornavirus, utilizes synonymous codon mutations to influence its evolution, resulting in the adaptive evolution of the virus to adverse environments. The phylogenetic tree and Median-joining (MJ)-Network of these SVA lineages worldwide were constructed to reveal SVA three-stage genetic development clusters. Furthermore, we analyzed the codon bias of the SVA genome of selected strains and found that SVA could increase the GC content of the third base of some amino acid synonymous codons to enhance the viral RNA adaptive evolution. Our results highlight the impact of recessive mutation of virus codon bias on the evolution of the SVA and uncover a previously underappreciated evolutionary strategy for SVA. They also underline the importance of understanding the genetic evolution of SVA and how SVA adapts to the adverse effects of external stress.

Seroprevalence of pullorum disease in chicken across mainland China from 1982 to 2020: A systematic review and meta-analysis.

Pullorum disease (PD), caused by the bacterium Salmonella pullorum, severely threatens the health of chickens worldwide, especially in China, and generating concerns for public health safety. Greater awareness of the seroprevalence may facilitate the prevention and control of this disease. We conducted systematic review and meta-analysis on the seroprevalence of PD in chicken flocks across mainland China. The results show that the overall pooled estimates of PD seroprevalence in chicken flocks was 18.2%. Furthermore, during 38-year period the seroprevalence of PD was markedly high in all seven regions, being at least 14.9% in central China. Our results suggest PD was highly prevalent in autumn, followed by winter. Chickens older than 120 days (22.6%, CI95: 14.5%-31.9%) had a significantly higher positive rate of PD than those <120 days in age (9.4%, CI95: 3.7%-17.4%). Additionally, the rearing mode used is a risk factor associated with the seroprevalence of PD, it being considerably lower for caged chickens (13.7%, CI95: 7.1%-22.0%) than free-range chickens (30.4%, CI95: 17.3-45.4%). Our findings demonstrate that PD still poses a major threat to poultry industries in mainland China, and therefore comprehensive and stringent strategies are needed to prevent and control this disease.

Global Infection Rate of Rotavirus C during 1980-2022 and Analysis of Critical Factors in the Host Range Restriction of Virus VP4.

Information on rotavirus C (RVC) infection is lacking, partly because the prevalence of RVC among humans and animals worldwide is undefined. Data on the characteristics of the P genotype among RVC strains are also required. We performed systematic searches on the infection rates of RVC since 1980 based on the literature and gene sequences of the PubMed and GenBank databases. A phylogenetic tree of VP4 genes was constructed to evaluate the distribution of the P genotype of RVC from various hosts. The specific mutation motifs in VP8* with P [2]/P [4]/P [5] specificity were analyzed to elucidate their roles in host range restriction. The rate of RVC infection in humans has fallen from 3% before 2009 to 1%, whereas in animals it has risen from 10% to 25%. The P genotype of RVC showed strict host species specificity, and current human RVC infections are exclusively caused by genotype P [2]. In the VP8* hemagglutinin domain of the P [4]/P [5] genotype of swine RVC, specific insertion or deletion were found relative to the human P [2] genotype, and these motifs are a possible critical factor for host range restriction. Our findings highlight the need for further epidemiological surveillance, preventive strategies, and elucidation of the factors involved in the specific host range restriction of RVC-circulating strains.

Prevalence of subclinical mastitis among dairy cattle and associated risks factors in China during 2012-2021: A systematic review and meta-analysis.

Bovine mastitis, especially subclinical mastitis (SCM), is one of the most prevalent and economically costly diseases in the dairy industry worldwide. Understanding the prevalence and spatial distribution of bovine SCM and its associated risk factors will facilitate the prevention and control of the disease. We reviewed published studies pertaining to epidemiological surveys of SCM among dairy cows during the past decade (2012-2021) in China from inception to March 20, 2020, with PubMed, Clinical Trial, VIP, CNKI and databases being used to identify English and Chinese articles. Therefore, we retrieved 41 studies related to epidemiological surveys of SCM among dairy cows, using our eligibility criteria. We demonstrated that the prevalence of SCM in dairy cows was 37.7% during the selected periods, indicating a slight increase in the incidence of SCM in a comparison between 2012 and 2016 and 2017-2021. The estimated prevalence of SCM was 36.4%-50.2% in the seven regions, which was no statistically significant difference. The highest prevalence of SCM was 72% in Inner Mongolia Autonomous Region, and the lowest prevalence was 19% in Hubei Province. The variation in diagnostic methods was not significant in the studies. Dairy cows' parity was a risk factor associated with the prevalence rate of SCM. Cow's age might affect the prevalence of SCM during the selected periods. This study may facilitate the control with specific strategies to reduce costs and antibiotics overuse, enhance food safety and public health.

Enhanced immunogenicity of foot and mouth disease DNA vaccine delivered by PLGA nanoparticles combined with cytokine adjuvants.

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P < 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.

[Cloning, expression and immunogenicity analysis of cathepsin L-like protease of Fasciola hepatica].

OBJECTIVE: To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein. METHODS: Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis. RESULTS: PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL. CONCLUSION: The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.

Mapping a highly conserved linear neutralizing epitope on gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae, causes a variety of diseases, which result in significant economic losses worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an important role in viral entry into the permissive cells, and protective immune response. The fine mapping epitope on the gD will contribute to the understanding of viral pathogenesis and development of alternative vaccines against various diseases associated with BoHV-1. We previously reported the preparation of a monoclonal antibody (MAb) 2B6, which was raised by a truncated recombinant gD protein, demonstrating a neutralizing activity against BoHV-1 infection in Madin-Darby bovine kidney cells. This study described the identification of a linear B-cell epitope on gD using MAb 2B6. A series of partially overlapping gD proteins with glutathione S-transferase tag were generated to define the epitope recognized by MAb 2B6. The amino acid (aa) sequence 323GEPKPGPSPDADRPE337 was recognized by MAb 2B6 using Western blot with the variedly truncated recombinant proteins. Importantly, this epitope was highly conserved among the typical members of BoHV-1, indicating that the epitope may be utilized in diagnosis of diseases due to BoHV-1 infection. Furthermore, the minimal linear epitope sequence 323GEPKPGP329 on gD recognized by MAb 2B6 was confirmed using single-aa residue deletion mutation in carboxyl terminal. This finding not only contributes to our understanding of gD of BoHV-1 virion but also shows a potential for the development of vaccine candidates and diagnostic techniques.

Brucellosis seroprevalence in dairy cattle in China during 2008-2018: A systematic review and meta-analysis.

Brucellosis remains one of the most common zoonotic diseases globally with more than a half million human cases reported annually. The Brucella reservoir associated with livestock brucellosis poses a significant threat to public health, and awareness of the seroprevalence and spatial distribution of livestock brucellosis is valuable for the prevention and control of diseases caused by Brucella, especially human brucellosis. Therefore, we conducted a systematic review and meta-analysis to evaluate the seroprevalence of brucellosis in dairy cattle in China. We retrieved 88 studies related to the seroprevalence of brucellosis in dairy cattle in China in which samples were harvested between 2008 and 2018. The results of our systematic review and meta-analysis reveal that the overall seroprevalence of brucellosis in dairy cattle herds in China was 1.9% during the selected period, rising from 1.6% in 2008-2012 to 2.6% in 2013-2018. In Northern China, where the traditional agropastoral areas with more developed animal breeding industry are located, the brucellosis seroprevalence was >10%. In contrast, the seroprevalence of brucellosis in Southern China reached only 5.5%. At the provincial level, the highest brucellosis seroprevalence in dairy cattle was estimated at >30% in Jilin province, followed by Shanxi, Ningxia, Inner Mongolia, and Guizhou, each with a prevalence of 10-20%. Additionally, the seroprevalence of brucellosis in some local areas was >30% or even >50%, indicating that Brucella infection was highly endemic in dairy herds in China. Our data may facilitate the prevention and control of brucellosis in domestic animals in China. Further epidemiological surveillance and the administration of a comprehensive monitoring program to determine the risk factors associated with brucellosis incidence in humans and domestic animals are recommended to refine brucellosis control strategies.

A systematic review and meta-analysis of the epidemiology of bovine viral diarrhea virus (BVDV) infection in dairy cattle in China.

Bovine viral diarrhea virus (BVDV) infection causes significantly economic losses to cattle industry worldwide, also including China. The epidemiological prevalence of infection associated with BVDV in dairy cattle has not been systematically assessed in China. Therefore, we undertook this study to evaluate prevalent of BVDV infection. We conducted a systematic review and meta-analysis of data from papers on the BVDV incidence and prevalence in dairy cattle in China by searching China Science and Technology Journal Database, China National Knowledge Infrastructure (CNKI), Wan Fang Database and PubMed for publication from March 2003 to March 2018. The 41 studies reporting the prevalence of BVDV in cattle in China were selected upon our inclusion criterion. The pooled BVDV prevalence in dairy cattle in China was estimated to 53.0% (95% CI 40.2-65.7) based on the data obtained from the 27,530 cows tested using serological or virological assay in the qualified papers published during the periods (χ² = 51,861.0, I2 = 99.9%). The highest BVDV positive rate in dairy flocks reached 90.0% in Fujian province of China, followed by Shaanxi (88.9%) and Shandong (83.3%). The prevalence in the six administrative districts of China was validated to be highly variable (25.7%-72.2%) and reached 72.2% in dairy cattle flocks of Northern China. Besides, the BVDV-RNA positive rate was estimated 27.1% (95% CI 17.3-37.0) based on 6 studies, comparatively, the pooled BVDV seroprevalence based on 35 studies was about 57.0% (95% CI 44.4-69.5) in China. This systematic review and meta-analysis firstly established an estimated prevalence of BVDV in dairy herds in China, indicating that the BVDV infection is escalating, though there is a bias in the number of studies between 2003-2009 and 2010-2018 timescales. This study may help understand the status of BVDV infection in dairy herds in China. Further extensive and comprehensive investigation is recommended, and effective intervention measures for preventing and controlling BVDV spread in dairy herds should be deployed, especially herds that have been exposed to BVDV.

Preparation of a Monoclonal Antibody Against gD Protein of Bovine Herpesvirus I.

The two DNA fragments encoding predicted main antigenic regions, aa 20-160 and aa 257-344 on gD protein of bovine herpesvirus-1 (BoHV-1) were tandem-cloned into the vector pET28a. The recombinant His-tagged Δ gD1-Δ gD2 was expressed in Escherichia coli BL21(DE3)pLysS by induction with 0.5 mM isopropyl-1-thio-ß-D-galactoside (IPTG) and Western blot analysis demonstrated that the recombinant His-tagged Δ gD1-Δ gD2 reacted with the positive serum against BoHV-1. A monoclonal antibody (MAb), designated as 2B6, was prepared by fusion of SP2/0 myeloma cells with splenocytes from a female 8-week-old BALB/c mouse immunized with purified His-tagged Δ gD1-Δ gD2 protein with the addition of Freund's adjuvant. The titer of the ascitic fluid triggered by hybridoma cells secreting MAb 2B6 was 1:2.5 × 108 and the subtype of MAb 2B6 was IgG 2a/κ. MAb 2B6 positively recognized with BoHV-1 infected Madin-Darby bovine kidney cells by an indirect fluorescent antibody assay. Moreover, MAb 2B6 showed 1:160 viral neutralizing activity using 60% plaque reduction assay. Therefore, this work suggested that MAb 2B6 has potential in the diagnosis of bovine respiratory disease complex associated with BoHV-1 and function analysis.