Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Detection of Escherichia coli O157H7 and Campylobacter jejuni in Bovine Carcasses in Two Slaughterhouses in Bio-Bío District, Chile.

Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Antimicrobial and antibiofilm effect of cannabinoids from Cannabis sativa against methicillin-resistant Staphylococcus aureus (MRSA) causing bovine mastitis.

Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml-1 with 86% mortality. At a sub-MIC dosage of 0.125 mg ml-1, the biofilm formation was reduced by 71%. The two major cannabinoids detected were cannabidiol and delta-9-tetrahydrocannabinol (Δ9-THC), which were majorly attributed to substantial inhibitory action against MRSA. The time-kill kinetics demonstrated a bactericidal action at 4 MIC over an 8-20-h time window with a 90% reduction in growth rate. The results from SEM, and light microscopy Giemsa staining revealed a reduction in cells in the treated group with increased AKP activity, indicating bacterial cell membrane breakdown. These findings suggested cannabinoids may be a promising alternative to antibiotic therapy for bovine biofilm-associated MRSA.

Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Insulin interacts with PPARγ agonists to promote bovine adipocyte differentiation.

Insulin is a potent adipogenic hormone that triggers a series of transcription factors that regulate the differentiation of preadipocytes into mature adipocytes. Ciglitazone specifically binds to peroxisome proliferator-activated receptor-γ (PPARγ), thereby promoting adipocyte differentiation. As a natural ligand of PPARγ, oleic acid (OA) can promote the translocation of PPARγ into the nucleus, regulate the expression of downstream genes, and promote adipocyte differentiation. We hypothesized that ciglitazone and oleic acid interact with insulin to enhance bovine preadipocyte differentiation. Preadipocytes were cultured 96 h in differentiation medium containing 10 mg/L insulin (I), 10 mg/L insulin + 10 µM cycloglitazone (IC), 10 mg/L insulin + 100 µM oleic acid (IO), or 10 mg/L insulin + 10 µM cycloglitazone+100 µM oleic acid (ICO). Control preadipocytes (CON) were cultured in differentiation medium (containing 5% fetal calf serum). The effects on the differentiation of Yanbian cattle preadipocytes were examined using molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. I, IC, IO, and ICO treatments produced higher concentrations of triglycerides (TAG) and lipid droplet accumulation in preadipocytes compared with CON treatment (P < 0.05). Co-treatment of insulin and PPARγ agonists significantly increased the expression of genes involved in regulating adipogenesis and fatty acid synthesis. (P < 0.05). Differential expression analysis identified 1488, 1764, 1974 and 1368 DEGs in the I, IC, IO and ICO groups, respectively. KEGG pathway analysis revealed DEGs mainly enriched in PPAR signalling, FOXO signaling pathway and fatty acid metabolism. These results indicate that OA, as PPARγ agonist, can more effectively promote the expression of bovine lipogenesis genes and the content of TAG and adiponectin when working together with insulin, and stimulate the differentiation of bovine preadipocytes. These findings provide a basis for further screening of relevant genes and transcription factors in intramuscular fat deposition and meat quality to enhance breeding programs.

Comparison of two serological diagnosis tests for bovine paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.

Molecular prevalence and associated risk factors of Cryptosporidium spp. infection in dairy cattle in Khon Kaen, Thailand.

Background and Aim: Cryptosporidium spp. are important parasites in the small intestines of humans and animals, particularly cattle. The aim of this study was to estimate the molecular prevalence and associated risk factors of Cryptosporidium infection in dairy cattle in five districts of Khon Kaen province, Thailand, and to identify Cryptosporidium spp. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from three groups of dairy cattle: Calves aged <3 months, calves aged 3 months-1 year, and calves aged >1 year. Cryptosporidium spp. were detected by polymerase chain reaction (PCR) amplifying the 18s RNA gene. Both genus-specific and species-specific primers were used to identify Cryptosporidium confirmed by DNA sequencing. Age, house floor type, and water trough type were evaluated as risk factors. We analyzed all associated risk factor information using the logistic regression test in the Statistical Package for the Social Sciences. Results: PCR results showed that 40 (13.51%) out of 296 samples were positive for Cryptosporidium spp., including Cryptosporidium bovis (57.50%) and Cryptosporidium ryanae (2.50%). There was a significant association between Cryptosporidium incidence, cattle age, and house floor type (p < 0.05). National Center for Biotechnology Information Basic Local Alignment Search Tool displayed 99.48%-100% nucleotide similarity of each Cryptosporidium spp. isolate with references recorded on GenBank. Conclusion: C. bovis and C. ryanae are commonly found in dairy cattle, especially calves, in Khon Kaen, Thailand, and the incidence was associated with age and house floor type. A molecular technique may be influential for species identification. The results of the present study would provide useful information for veterinarians and animal owners to understand better Cryptosporidium spp. and how to manage farms properly.

First report on the molecular detection and genetic diversity of Anaplasma marginale in healthy dairy cattle in Khon Kaen province, Thailand.

Background and Aim: Bovine anaplasmosis (BA) is one of the most important diseases of ruminants worldwide, causing significant economic losses in the livestock industry due to the high morbidity and mortality in susceptible cattle herds. Anaplasma marginale is the main causative agent of BA occurring worldwide in tropical and subtropical regions. This study aimed to investigate the first molecular detection and genetic diversity of A. marginale in dairy cattle in Khon Kaen Province, Thailand. Materials and Methods: Blood samples were collected from 385 lactating cows from 40 dairy farms in five districts of Khon Kaen, regardless of age and health status. To detect A. marginale, all DNA preparations were used for molecular diagnosis using a single polymerase chain reaction with the msp4 gene target. A phylogenetic tree was constructed from the msp4 gene sequences using molecular genetic characterization. Genetic diversity was calculated as haplotype diversity, haplotype number, number of nucleotide differences, nucleotide diversity, and average number of nucleotide differences. Results: The overall prevalence of A. marginale was 12.72% (49/385). The highest prevalence (17.19%) was found in Ubolratana district, followed by Muang, Kranuan, Khao Suan Kwang, and Nam Phong districts (14.94%, 14.74%, 13.79%, and 3.70%, respectively). Phylogenetic analysis showed that A. marginale was closely related to isolates from Australia (98.96%), China (99.68%), Spain (99.74%), and the USA (99.63%). Conclusion: The molecular prevalence of BA in dairy cattle is the first to be observed in this area, and the genetic variability with separated clusters shown in the msp4 gene of A. marginale revealed species variation in dairy cattle. This significant genetic diversity contributes to the understanding of the diversity of A. marginale and will be important for the control and prevention of A. marginale in dairy cattle.

The role, relevance and management of immune exhaustion in bovine infectious diseases.

Immune exhaustion is a state of immune cell dysfunction that occurs most commonly following chronic exposure to an antigen which persists after the immune response fails to remove it. Exhaustion has been studied most thoroughly with several cancers, but has also been observed in several chronic infectious diseases. The topic has mainly been studied with CD8+ T cells, but it can also occur with CD4+ T cells and other immune cell types too. Exhaustion is characterized by a hierarchical loss of effector cell functions, up-regulation of immuno-inhibitory receptors, disruption of metabolic activities, and altered chromatin landscapes. Exhaustion has received minimal attention so far in diseases of veterinary significance and this review's purpose is to describe examples where immune exhaustion is occurring in several bovine disease situations. We also describe methodology to evaluate immune exhaustion as well as the prospects of controlling exhaustion and achieving a more suitable outcome of therapy in some chronic disease scenarios.

TREATMENT OF AORTO-ILIAC AND INFRA-INGUINAL VASCULAR INFECTIONS WITH A PREFABRICATED BOVINE PERICARDIAL GRAFT.

OBJECTIVE: The use of biological grafts provides acceptable mid- and long-term results in native or prosthetic vascular infections. Several reports describe the successful use of bovine pericardium in case of vascular infections, mainly as a large patch to be sutured as a tubular graft. Recently, a novel prefabricated bovine pericardium graft (Biointegral Surgical No-React® Inc, Mississauga, ON, Canada) has been introduced in clinical practice with promising results. In this study, we report our preliminary experience utilizing Biointegral Surgical graft in case of native and or prosthetic aorto-iliac and infra-inguinal infection. METHODS: We retrospectively analyzed data from 20 patients with native or prosthetic aorto-iliac and infra-inguinal infection who underwent in situ reconstruction with a Biointegral Surgical No-React bovine pericardium prosthesis between October 2020 and February 2023 at the Vascular Surgery Unit of the Fondazione Policlinico Universitario Gemelli - IRCCS in Rome, Italy. All patients followed a standardized protocol including postoperative anticoagulation and long-term intravenous antibiotics. RESULTS: The indication for surgery was: mycotic aortic aneurysm in four patients (20%), graft infection after abdominal aortic repair in 11 patients (55%), peripheral graft infection in five patients (25%). Complete excision of the infected aorta or prosthetic graft, surgical debridement and in situ reconstruction were performed in all patients. Hospital mortality rate was 5% (n=1) and graft-related mortality of 0%. During follow-up (median 13 months, range 6-34 months), reinfection was 5.2% and primary graft patency 94.7%. CONCLUSIONS: The use of prefabricated bovine pericardial grafts represent a promising option for the treatment of native and prosthetic aorto-iliac and infra-inguinal infections. The application of this biological graft with a standardized post-operative protocol has been associated with a satisfactory patency and reinfection rate without increased bleeding complications.

Exploring the interaction of a potent anti-cancer drug Selumetinib with bovine serum albumin: Spectral and computational attributes.

The binding of drugs to plasma proteins determines its fate within the physiological system, hence profound understanding of its interaction within the bloodstream is important to understand its pharmacodynamics and pharmacokinetics and thereby its therapeutic potential. In this regard, our work delineates the mechanism of interaction of Selumetinib (SEL), a potent anti-cancer drug showing excellent effect against multiple solid tumors, with plasma protein bovine serum albumin (BSA), using methods such as absorption, steady-state fluorescence, time-resolved, fluorescence resonance energy transfer, Fourier transform infrared spectra (FTIR), circular dichroism (CD), synchronous and 3D-fluorescence, salt fluorescence, molecular docking and molecular dynamic simulations. The BSA fluorescence intensity was quenched with increasing concentration of SEL which indicates interactions of SEL with BSA. Stern-Volmer quenching analysis and lifetime studies indicate the involvement of dynamic quenching. However, some contributions from the static quenching mechanism could not be ruled out unambiguously. The association constant was found to be 5.34 × 105 M-1 and it has a single binding site. The Förster distance (r) indicated probable energy transmission between the BSA and SEL. The positive entropy changes and enthalpy change indicate that the main interacting forces are hydrophobic forces, also evidenced by the results of molecular modeling studies. Conformation change in protein framework was revealed from FTIR, synchronous and 3D fluorescence and CD studies. Competitive binding experiments as well as docking studies suggest that SEL attaches itself to site I (subdomain IIA) of BSA where warfarin binds. Molecular dynamic simulations indicate the stability of the SEL-BSA complex. The association energy between BSA and SEL is affected in the presence of different metals differently.

Bovine lactoferrin inhibits inflammatory response and apoptosis in lipopolysaccharide-induced acute lung injury by targeting the PPAR-γ pathway.

BACKGROUND: Lactoferrin (LF) is an iron-binding multifunctional cationic glycoprotein. Previous studies have demonstrated that LF may be a potential drug for treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In this study, we explored the anti-inflammatory effect and mechanism of bovine lactoferrin (bLF) in ALI using the RNA sequencing (RNA-seq) technology and transcriptome analysis. METHODS AND RESULTS: Based on the differentially expressed genes (DEGs) obtained from RNA-seq of the Lung from mouse model, the bioinformatics workflow was implemented using the BGISEQ-500 platform. The protein-protein interaction (PPI) network was obtained using STRING, and the hub gene was screened using Cytoscape. To verify the results of transcriptome analysis, the effects of bLF on Lipopolysaccharide (LPS)-induced BEAS-2B cells and its anti-reactive oxygen species (ROS), anti-inflammatory, and antiapoptotic effects were studied via Cell Counting Kit-8 (CCK-8) test, active oxygen detection test, ELISA, and western blot assay. Transcriptome analysis revealed that two hub gene modules of DEGs were screened via PPI analysis using the STRING and MCODE plug-ins of Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these core modules are enriched in the PPAR (peroxisome proliferator-activated receptor) and AMPK (AMP-activated protein kinase) signaling pathways. Through cell experiments, our study shows that bLF can inhibit ROS, inflammatory reaction, and LPS-induced BEAS-2B cell apoptosis, which are significantly antagonized by the PPAR-γ inhibitor GW9662. CONCLUSION: This study has suggested that the PPAR-γ pathway is the critical target of bLF in anti-inflammatory reactions and apoptosis of ALI, which provides a direction for further research.

Insights on the dynamic behavior of protein disulfide isomerase in the solution environment through the SAXS technique.

The dynamic behavior of Protein Disulfide Isomerase (PDI) in an aqueous solution environment under physiologically active pH has been experimentally verified in this study using Small Angle X-ray Scattering (SAXS) technique. The structural mechanism of dimerization for full-length PDI molecules and co-complex with two renowned substrates has been comprehensively discussed. The structure models obtained from the SAXS data of PDI purified from bovine liver display behavior duality between unaccompanied-enzyme and after engaged with substrates. The analysis of SAXS data revealed that PDI exists as a homo-dimer in the solution environment, and substrate induction provoked its segregation into monomer to enable the enzyme to interact systematically with incoming clients. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00198-0.

Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases.

Bovine respiratory disease (BRD) is a significant veterinary challenge, often exacerbated by pathogen resistance, hindering effective treatment. Traditional testing methods for primary pathogens - Mycoplasma bovis, Pasteurella multocida, and Mannheimia haemolytica - are notably time-consuming and lack the rapidity required for effective clinical decision-making. This study introduces a TaqMan MGB probe detection chip, utilizing fluorescent quantitative PCR, targeting key BRD pathogens and associated drug-resistant genes and sites. We developed 94 specific probes and primers, embedded into a detection chip, demonstrating notable specificity, repeatability, and sensitivity, reducing testing time to under 1 h. Additionally, we formulated probes to detect mutations in the quinolone resistance-determining region, associated with fluoroquinolone resistance in BRD pathogens. The chip exhibited robust sensitivity and specificity, enabling rapid detection of drug-resistant mutations in clinical samples. This methodology significantly expedites the diagnostic process for BRD and sensitive drug screening, presenting a practical advancement in the field.

A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study's protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. KEY POINTS: • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

Hesperidin ameliorates H2O2-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway.

BACKGROUND: Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells (bMECs) exposed to oxidative stress have not been elucidated. RESULTS: In this study, we investigated the effects of hesperidin on H2O2-induced oxidative stress in bMECs and the underlying molecular mechanism. We found that hesperidin attenuated H2O2-induced cell damage by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, increasing catalase (CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent. CONCLUSIONS: Our results suggest that hesperidin could protect bMECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Design of a multi-epitope-based vaccine candidate against Bovine Genital Campylobacteriosis using a reverse vaccinology approach.

BACKGROUND: Bovine Genital Campylobacteriosis (BGC), a worldwide distributed venereal disease caused by Campylobacter fetus subsp. venerealis (Cfv), has a relevant negative economic impact in cattle herds. The control of BGC is hampered by the inexistence of globally available effective vaccines. The present in silico study aimed to develop a multi-epitope vaccine candidate against Cfv through reverse vaccinology. RESULTS: The analysis of Cfv strain NCTC 10354 proteome allowed the identification of 9 proteins suitable for vaccine development. From these, an outer membrane protein, OmpA, and a flagellar protein, FliK, were selected for prediction of B-cell and T-cell epitopes. The top-ranked epitopes conservancy was assessed in 31 Cfv strains. The selected epitopes were integrated to form a multi-epitope fragment of 241 amino acids, which included 2 epitopes from OmpA and 13 epitopes from FliK linked by GPGPG linkers and connected to the cholera toxin subunit B by an EAAAK linker. The vaccine candidate was predicted to be antigenic, non-toxic, non-allergenic, and soluble upon overexpression. The protein structure was predicted and optimized, and the sequence was successfully cloned in silico into a plasmid vector. Additionally, immunological simulations demonstrated the vaccine candidate's ability to stimulate an immune response. CONCLUSIONS: This study developed a novel vaccine candidate suitable for further in vitro and in vivo experimental validation, which may become a useful tool for the control of BGC.

Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Factors associated with perceived Anaplasma marginale infection and clinical anaplasmosis cases on beef operations in California.

Bovine anaplasmosis is a tick-borne disease caused by Anaplasma marginale in the United States. The objective of this study was to use a survey tool to generate information for beef operations in California on anaplasmosis prevention and control management, including to what extent management activities were informed by perceived herd-level exposure to A. marginale infection or occurrence of clinical anaplasmosis cases. We mailed 2,621 questionnaires with questions on Anaplasma status, herd demographics, anaplasmosis control and prevention measures, and environmental factors to beef ranchers in California in October 2020. Survey-weighted chi-square tests were used to compare management differences according to perceived Anaplasma infection status. Generalized estimating equations (GEEs) were used to analyze whether region of California, management practices, or environmental factors were associated with reported clinical cases of anaplasmosis in the previous five years. A total of 466 questionnaires describing 749 herds were obtained and used in this study. Use of management measures, including deliberate exposure of calves to ticks, vaccination for Anaplasma, infection control through antibiotics in feed, maintaining a completely closed herd, blood testing for Anaplasma on all herd additions, and taking no anaplasmosis control and prevention measures, were significantly different between herds with or without perceived A. marginale infection based on producers' self-declared status. The overall perceived prevalence for Anaplasma infection and reported clinical cases of anaplasmosis at the herd level was 26.0 % (95 % CI: 24.3-27.7 %) and 17.1 % (95 % CI: 15.6-18.6 %) respectively, with the highest perceived infection and case numbers reported in the Central Coast region. In the GEE model, higher odds of reporting clinical cases of anaplasmosis in the previous five years were observed in cattle located in the Central Coast region, cattle within a large herd, cattle that are treated with tick/fly control, cattle in a completely closed herd, and cattle receiving Anaplasma vaccine. Anaplasma infection and bovine anaplasmosis status may be underestimated in beef herds in California based on previous study results. Changing needles between cattle after injections and conducting blood testing for Anaplasma on herd additions are important Anaplasma management measures that are infrequently implemented in beef herds in California. The results show a need for producer education to improve producers' awareness of bovine anaplasmosis and implement proper measures for disease control and prevention.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.