Canine parvovirus 2 (CPV-2) is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies. The genetic variability and antigenic diversity of CPV-2 hinder its effective prevention of infection by vaccination. To investigate the epidemiology and genetic characteristics of CPV-2 in China, rectal swabs from affected dogs were collected from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and capsid gene sequencing of CPV-2 were performed using previously described primers and protocols. The overall detection rate for CPV-2 was 16.5% (33/200). A significant association was found between the CPV-2-positivity and clinical signs, age, breed and vaccination status. Sequence analysis revealed the presence of CPV-2c genotypes in all positive samples, which were genetically similar to other Asian CPV-2c strains. Notably, four key mutations (A5G, F267Y, Y324I and Q370R) were detected in all isolates, and one novel mutation (I447M) was detected in three CPV-2 isolates. These mutations in the CPV-2 strains could impact vaccine efficacy and the effectiveness of the virus immune evasion. Surprisingly, no recombination events were observed between the identified CPV-2c strains and reference strains from China. Our data revealed that amino acid residues 324, 426 and 440 of VP2 may under strong selection pressure. This pattern of genetic variation in the CPV-2 lineage warrants continuous laboratory-based surveillance programs in other parts of China to better understand the pattern of seasonal distribution and association between emerging genotypes and the intensity of disease severity.
As a new data management paradigm, knowledge graphs can integrate multiple data sources and achieve quick responses, reasoning and better predictions in drug discovery. Characterized by powerful contagion and a high rate of morbidity and mortality, porcine reproductive and respiratory syndrome (PRRS) is a common infectious disease in the global swine industry that causes economically great losses. Traditional Chinese medicine (TCM) has advantages in low adverse effects and a relatively affordable cost of application, and TCM is therefore conceived as a possibility to treat PRRS under the current circumstance that there is a lack of safe and effective approaches. Here, we constructed a knowledge graph containing common biomedical data from humans and Sus Scrofa as well as information from thousands of TCMs. Subsequently, we validated the effectiveness of the Sus Scrofa knowledge graph by the t-SNE algorithm and selected the optimal model (i.e., transR) from six typical models, namely, transE, transR, DistMult, ComplEx, RESCAL and RotatE, according to five indicators, namely, MRR, MR, HITS@1, HITS@3 and HITS@10. Based on embedding vectors trained by the optimal model, anti-PRRSV TCMs were predicted by two paths, namely, VHC-Herb and VHPC-Herb, and potential anti-PRRSV TCMs were identified by retrieving the HERB database according to the pharmacological properties corresponding to symptoms of PRRS. Ultimately, Dan Shen's (Salvia miltiorrhiza Bunge) capacity to resist PRRSV infection was validated by a cell experiment in which the inhibition rate of PRRSV exceeded 90% when the concentrations of Dan Shen extract were 0.004, 0.008, 0.016 and 0.032 mg/mL. In summary, this is the first report on the Sus Scrofa knowledge graph including TCM information, and our study reflects the important application values of deep learning on graphs in the swine industry as well as providing accessible TCM resources for PRRS.
Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic CoV that causes severe vomiting, diarrhea and dehydration in suckling piglets, leading to economic losses in the swine industry. There is a great need for a convenient method to detect circulating antibodies and help in accurate diagnosis and disease control. Previously, we demonstrated that a unique PDCoV accessory protein, NS6, is expressed during PDCoV infection in pigs and is incorporated into PDCoV virions; thus, we deduced that NS6 is likely an immunogenic target that can be used for the diagnosis of PDCoV infection. In this study, we first confirmed that NS6 is immunogenic in PDCoV-infected pigs by performing a serum western blot. Furthermore, we developed a novel NS6-based indirect enzyme-linked immunosorbent assay (iELISA) method and compared it to an established S1-based iELISA for the survey of anti-PDCoV IgG or IgA in pigs of different ages in China. The NS6-iELISA has high specificity for the detection of IgG antibodies and no cross-reactivity with other porcine enteric CoVs (transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, or swine acute diarrhea syndrome coronavirus). This NS6 serology-based method has great sensitivity and good repeatability, making it a new and cost-saving option for the rapid diagnosis and immunosurveillance of PDCoV, which may also be important for the prevention and control of deltacoronavirus-related infection in pigs and other animals.
The widespread occurrence of carbapenem-resistant organisms has garnered significant public attention. Arthropods, including flies, are important vectors of multidrug-resistant bacteria. In this study, we reported the simultaneous carriage of four carbapenem-resistant isolates from different species, namely, Escherichia coli (E. coli), Providencia manganoxydans (P. manganoxydan), Myroides odoratimimus (M. odoratimimus) and Proteus mirabilis (P. mirabilis), from a single fly in China. These isolates were characterized through antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, and bioinformatics analysis. M. odoratimimus showed intrinsic resistance to carbapenems. The mechanisms of carbapenem resistance in E. coli, P. manganoxydans, and P. mirabilis were due to the production of NDM-5, NDM-1 and NDM-1, respectively. Genetic context of the bla NDM genes in these three isolates varied. The bla NDM-5 gene in E. coli was located on an IncHI2/HI2A multidrug-resistant plasmid, which was conjugatively transferable. The bla NDM-1 gene in P. mirabilis resided on the pPM14-NDM_123k-like nonconjugative plasmid. The bla NDM-1 gene in P. manganoxydans was found in a nonconjugatively transferable, multidrug-resistant region. The results of this study enhance our understanding of the dissemination of carbapenem-resistant organisms and suggest the need for a more comprehensive approach to antibiotic resistance research encompassing humans, animals, and the environment.
Feline panleukopenia virus (FPV) is a single-stranded DNA virus that can infect cats and cause feline panleukopenia, which is a highly contagious and fatal disease in felines. The sequence of FPV is highly variable, and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence, immunogenicity, host selection, and other abilities. In this study, the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifically, diarrhea or vomiting during the period spanning from 2018 to 2022. The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%. Capsid (virion) protein 2 (VP2) gene of each FPV-positive sample was sequenced and amplified, yielding 65 VP2 sequences. Among them, six VP2 gene sequences were detected in the majority of the samples test positive for FPV, and these positive samples originated from a diverse range of geographical locations. These isolates were named FPV-6, FPV-10, FPV-15, FPV-251, FPV-271 and FPV-S2. Additionally, the substitution of Ala300Pro (A300P) in VP2 was detected for the first time in feline-derived FPV (FPV-251). FPV-251 isolate, with this substitution in VP2 protein, exhibited stable proliferative capacity in Madin-Darby canine kidney (MDCK) cells and A72 cells. FPV-271 was selected as the FPV control isolate due to its single amino acid difference from VP2 protein of FPV-251 at position 300 (FPV-271 has alanine, while FPV-251 has proline). After oral infection, both FPV-251 and FPV-271 isolates caused feline panleukopenia, which is characterized by clinical signs of enterocolitis. However, FPV-251 can infect dogs through the oral route and cause gastrointestinal (GI) symptoms with lesions in the intestine and mesenteric lymph nodes (MLNs) of infected dogs. This is the first report on the presence of an A300P substitution in VP2 protein of feline-derived FPV. Additionally, FPV isolate with a substitution of A300P at VP2 protein demonstrated efficient replication capabilities in canine cell lines and the ability to infect dogs.
Trypanosoma cruzi is the etiologic agent of Chagas disease. This flagellated protozoan is transmitted to humans as well as different species of domestic and wild animals via vectors from the Reduviidae family (known as “kissing bugs”). Despite the fact that hundreds of species of wild mammals are part of the reservoir system, the morphological changes and clinical manifestations resulting from the pathogenesis of the infection have been largely neglected. The aim of this review is to systematically compile the available information regarding clinicopathological alterations in wild mammals due to natural infection by T. cruzi. Information was obtained from six online bibliographic data search platforms, resulting in the identification of 29 publications that met the inclusion criteria. Mortality was the most common clinical manifestation, cardiac damage was the main finding at necropsy, and lymphoplasmacytic inflammation was the most frequent microscopic injury. Thus, regardless of its role as a reservoir, T. cruzi has the potential to affect the health status of wild mammals, a situation that highlights the need for further research to analyze, measure, and compare its effects at both the individual and population levels.
Enterotoxigenic E. coli is one of the bacterial pathogens contributing to the global resistance crisis in public health and animal husbandry. The problem of antibiotic resistance is becoming more and more serious, and phage is considered one of the potential alternatives to antibiotics that could be utilized to treat bacterial infections. Our study isolated and identified a lytic phage PGX1 against multidrug-resistant enterotoxigenic E. coli EC6 strain from sewage. The phage lysis profile revealed that PGX1 exhibited a lytic effect on multidrug-resistant enterotoxigenic E. coli strains of serotype O60. Through phage whole genome sequencing and bioinformatics analysis, PGX1 was found to be the class Caudoviricetes, family Autographiviridae, genus Teseptimavirus. The length of the PGX1 genome is about 37,009 bp, containing 54 open reading frames (ORFs). Notably, phage PGX1 lacks any lysogenic-related genes or virulence genes. Furthermore, phage PGX1 demonstrates strong adaptability, tolerance, and stability in various pH (pH4-10) and temperatures (4–40°C). The in vivo and in vitro tests demonstrated that phage PGX1 significantly removes and inhibits the formation of multidrug-resistant EC6 biofilm and effectively controls the Galleria mellonella larvae and enterotoxigenic E. coli EC6 during mice infection. In conclusion, the above findings demonstrated that phage PGX1 may be a novel antimicrobial agent to control multidrug-resistant E. coli infections.
This is the first report on the screening, expression, and recognition mechanism analysis of single-chain fragment variable (scFv) against phenylethanolamine A (PEAA), a newly emerged β-adrenergic agonist illegally used as a feed additive for growth promotion. The PEAA-specific scFv scFv, called scFv-32, was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E. coli system. The ic-ELISA results revealed an IC50 value of 10.34 μg/L for scFv-32 and no cross-reactivity with other β-adrenergic agonists. Homology modeling and molecular docking revealed the key binding sites VAL178, TYP228, and ASP229. One hydrogen bond, two pi-sigma bonds, and one pi-pi bond maintain the formation of the antibody‒drug complex. Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity, which confirmed the accuracy of the theoretical analysis. These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes significant economic loss to the global pig industry. Genotype 1 and 2 PRRSV (PRRSV-1 and -2) infections have been reported in China, Europe and America. For accurate prevention, nanobodies were first used as diagnostic reagents for PRRSV typing. In this study three nanobodies targeting both PRRSV-1 and -2, two targeting PRRSV-1 and three targeting PRRSV-2, were screened and produced. To develop two competitive ELISAs (cELISAs), the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA, to detect common antibodies against PRRSV-1 and -2, and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA, to detect anti-PRRSV-1 antibodies. The two cELISAs were developed using PRRSV-1-N protein as coating antigen, and the amounts for both were 100 ng/well. The optimized dilution of testing pig sera was 1:20, the optimized reaction times were 30 min, and the colorimetric reaction times were 15 min. Then, the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6% and 35.6%, respectively. Both of them have high sensitivity, strong specificity, good repeatability, and stability. In addition, for the 1534 clinical pig sera, an agreement rate of 99.02% (Kappa values = 0.97) was determined between the g1-2-cELISA and the commercial IDEXX ELISA kit. For the g1-cELSIA, it can specifically detect anti-PRRSV-1 antibodies in the clinical pig sera. Importantly, combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and -2.
The rapid emergence and spread of colistin-resistant gram-negative bacteria has raised worldwide public health concerns, and phosphoethanolamine (PEtn) transferase modification-mediated colistin resistance has been widely documented in multiple gram-negative bacterial species. However, whether such a mechanism exists in the zoonotic pathogen Pasteurella multocida is still unknown. Recently, a novel PEtn transferase, PetL, was identified in P. multocida, but whether it is associated with colistin resistance remains to be elucidated. In this study, we found that PetL in P. multocida (PetLPM) exhibited structural characteristics similar to those of the mobile-colistin-resistant (MCR) protein and the PEtn transferase characterized in Neisseria meningitidis. The transformation of petL PM into E. coli or K. pneumoniae changed the phenotype of several tested strains from colistin sensitive to colistin resistant. Deletion of this gene decreased the colistin minimum inhibitory concentration (MIC) of P. multocida by 64-fold. Our extensive analysis by MALDI-TOF–MS demonstrated that PetLPM participated in the modification of bacterial lipopolysaccharide (LPS)-lipid A. Deletion of petL PM led to an increase in membrane charge but a decrease in cell-surface hydrophobicity and cell permeability in P. multocida. The present study is the first to report the presence of PEtn transferase-mediated colistin resistance in the zoonotic pathogen P. multocida.
Zoonotic hookworm infections remain a significant public health problem, causing nearly 500 million cases globally and approximately four million disability-adjusted life years lost annually. More than one-fifth of these cases are attributed to Ancylostoma ceylanicum, an emerging zoonotic health issue in the Asia-Pacific region. This review presents key research gaps regarding the epidemiology, diagnosis, control, prevention and elimination of A. ceylanicum and other canine zoonotic hookworms as neglected health threats. A. ceylanicum is the second most prevalent human hookworm in the region; it is the most common hookworm among dogs and cats-reservoirs of zoonotic infections. Previous population genetic and phylogenetic analyses revealed that A. ceylanicum has three possible transmission dynamics: zoonotic, animal-only, and human-only pathways. The actual burden of zoonotic ancylostomiasis in most endemic countries remains unknown due to the use of parasitological techniques (e.g., Kato-Katz thick smear and floatation techniques) that have reduced diagnostic performance and do not allow accurate species identification in helminth surveys. The emergence of benzimidazole resistance in soil-transmitted helminths (STHs), including hookworms, is a concern due to the protracted implementation of mass drug administration (MDA). Resistance is conferred by single nucleotide polymorphisms (SNPs) that occur in the β-tubulin isotype 1 gene. These mutations have been reported in drug-resistant A. caninum but have not been found in A. ceylanicum in the field. A. ceylanicum remains understudied in the Asia-Pacific region. The zoonotic nature of the parasite warrants investigation of its occurrence in human and animal reservoir hosts to understand the dynamics of zoonotic transmission in different endemic foci. The detection of benzimidazole resistance-associated SNPs in zoonotic hookworms from Asia-Pacific countries has yet to be thoroughly explored. Considering the high level of hookworm endemicity in the region, the circulation of resistant isolates between humans and animals potentially presents a significant One Health threat that can undermine current MDA and proposed animal deworming-based control efforts.
The development of alternative therapies to treat chicken coccidiosis has become a hot topic because of the widespread use of conventional medicines. This study aimed to investigate the effectiveness of eugenol in treating Eimeria tenella infection in broilers. Broiers, at the age of 14 d, were orally infected with sporulated Eimeria tenella oocysts, and then, eugenol essential oil was added to chicken feed at three different dosages (0.1, 0.2 or 0.4 g/kg). The anticoccidial effects of eugenol essential oil were assessed using the anticoccidial index (ACI). As a result, eugenol exhibited a moderate anticoccidial effect, with an ACI of 167.37 at 0.2 g/kg. After eugenol treatment, the expression of occludin in the epithelial cells of the chicken cecum was significantly greater (P < 0.05) than that in the epithelial cells of the nontreated control (IC) group. The proportion of intestinal Lactobacillus_agilli increased. Eugenol therapy dramatically increased the activity of superoxide dismutase. After high-dose treatment, the expression of the proinflammatory factors IL-1β and IL-6 significantly decreased, while the expression of the cytokines IL-4 and IFN-γ significantly increased. The safety of eugenol essential oil was evaluated at the 1, 3 or 6 recommended doses. Overall, no significant differences were detected in the blood tests or serum biochemistry of the chickens between the treatment groups and the control group. As a result, eugenol essential oil can cure chicken coccidiosis by improving the intestinal microbial structure in the chicken cecum and decreasing the cecum's inflammatory reactions, thus strengthening immune function and eventually demonstrating anticoccidial properties.
Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations. One promising area of research involves self-assembled protein nanoparticles (SAPNs), which have shown potential for enhancing antigen-presenting cell uptake, B-cell activation, and lymph node trafficking. Numerous nanovaccines have been utilized in veterinary medicine, including natural self-assembled protein nanoparticles, rationally designed self-assembled protein nanoparticles, animal virus-derived nanoparticles, bacteriophage-derived nanoparticles, and plant-derived nanoparticles, which will be discussed in this review. SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases. This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine. Furthermore, this paper highlights their disadvantages and methods for improving their immunogenicity.
Bovine coronavirus (BCoV) poses a significant threat to the global cattle industry, causing both respiratory and gastrointestinal infections in cattle populations. This necessitates the development of efficacious vaccines. While several inactivated and live BCoV vaccines exist, they are predominantly limited to calves. The immunization of adult cattle is imperative for BCoV infection control, as it curtails viral transmission to calves and ameliorates the impact of enteric and respiratory ailments across all age groups within the herd. This study presents an in silico methodology for devising a multiepitope vaccine targeting BCoV. The spike glycoprotein (S) and nucleocapsid (N) proteins, which are integral elements of the BCoV structure, play pivotal roles in the viral infection cycle and immune response. We constructed a remarkably effective multiepitope vaccine candidate specifically designed to combat the BCoV population. Using immunoinformatics technology, B-cell and T-cell epitopes were predicted and linked together using linkers and adjuvants to efficiently trigger both cellular and humoral immune responses in cattle. The in silico construct was characterized, and assessment of its physicochemical properties revealed the formation of a stable vaccine construct. After 3D modeling of the vaccine construct, molecular docking revealed a stable interaction with the bovine receptor bTLR4. Moreover, the viability of the vaccine’s high expression and simple purification was demonstrated by codon optimization and in silico cloning expression into the pET28a (+) vector. By applying immunoinformatics approaches, researchers aim to better understand the immune response to bovine coronavirus, discover potential targets for intervention, and facilitate the development of diagnostic tools and vaccines to mitigate the impact of this virus on cattle health and the livestock industry. We anticipate that the design will be useful as a preventive treatment for BCoV sickness in cattle, opening the door for further laboratory studies.
Bordetella bronchiseptica (Bb) is recognized as a leading cause of respiratory diseases in dogs and cats. However, epidemiological data on Bb in dogs and cats in China are still limited, and there is no commercially available vaccine. Live vaccines containing Bb that are widely used abroad are generally effective but can establish latency and potentially reactivate to cause illness in some immunodeficient vaccinated recipients, raising safety concerns. In this study, 34 canine-derived and two feline-derived Bb strains were isolated from 1809 canine and 113 feline nasopharyngeal swab samples collected from eight provinces in China from 2021 to 2023. The PCR results showed that the percentage of positive Bb was 22.94% (441/1922), and more than 90% of the Bb isolates had four virulence factor-encoding genes (VFGs), namely, fhaB, prn, betA and dnt. All the isolated strains displayed a multidrug-resistant phenotype. The virulence of 10 Bb strains isolated from dogs with respiratory symptoms was tested in mice, and we found that eight isolates were highly virulent. Furthermore, the eight Bb isolates with high virulence were inactivated and intramuscularly injected into mice, and three Bb strains (WH1218, WH1203 and WH1224) with the best protective efficacy were selected. Dogs immunized with these three strains exhibited strong protection against challenge with the Bb field strain WH1218. Ultimately, the WH1218 strain with the greatest protection in dogs was selected as the vaccine candidate. Dogs and cats that received a vaccine containing 109 CFU of the inactivated WH1218 strain showed complete protection against challenge with the Bb field strain WH1218. This study revealed that Bb is an important pathogen that causes respiratory diseases in domestic dogs and cats in China, and all the isolates exhibited multidrug resistance. The present work contributes to the current understanding of the prevalence, antimicrobial resistance, and virulence genes of Bb in domestic dogs and cats. Additionally, our results suggest that the WH1218 strain is a promising candidate safe and efficacious inactivated Bb vaccine.
The prevalence of bacterial digestive diseases in plateau animals has caused considerable losses to the Tibetan livestock industry. Therefore, this study aimed to isolate safe lactic acid bacteria (LAB) with beneficial probiotic properties to protect yaks from intestinal diseases. After 16S rDNA matching, four strains of Lactobacillus fermentum (A4), Pediococcus pentosaceus (A3.4 and A1.2), and Pediococcus acidilactici (B1.9) were isolated from the intestinal tissues and content of healthy yaks. The results indicated that A4 was more tolerant to bile salt (0.3%), while A3.4 had better stability in an acidic (pH = 3.0) environment. The results of the antibacterial activity test suggested that the isolates inhibited most pathogenic bacteria by up to 20%, except for A3.4, which inhibited Pasteurella and Staphylococcus aureus by more than 20%. Moreover, the results of the antioxidant test demonstrated that A4 and A3.4 had potent antioxidant activity. In addition, the drug sensitivity test revealed that the isolates were susceptible to commonly used antibiotics. In terms of safety, the isolates promoted growth, enhanced intestinal development, and protected the intestinal barrier without causing any adverse effects. In conclusion, LAB isolated from yak intestinal contents are potential probiotics with excellent antibacterial properties.
African swine fever (ASF), caused by the ASF virus (ASFV), is an acute, severe, and highly contagious infectious disease in domestic pigs and wild boars. Domestic pigs infected with a virulent ASFV strain can have morbidity and mortality rates of up to 100%. The epidemic of ASF has caused serious economic losses to the global pig industry. Currently, there is no safe and effective vaccine or specific drug for treating ASF. Therefore, ASFV still poses a great threat to pig factories. ASFV is a double-stranded DNA virus with a complex icosahedral multilayer structure. The ASFV genome contains 150–170 open reading frames (ORFs) that encode 150–200 proteins. Some ASFV-encoded proteins are involved in virus invasion, genome replication, DNA repair, and virion formation. Some ASFV proteins execute immunomodulatory functions by regulating the host antiviral innate immune response. Accumulating studies have shown that the immunomodulatory functions of ASFV genes are closely related to the virulence and pathogenicity of ASFV isolates. This review summarizes the research advances on ASFV immune evasion mechanisms in African swine fever patients and provides new insights for developing attenuated live vaccine candidates to prevent and control ASF.
African swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by African swine fever virus (ASFV). The mortality rate associated with ASF is remarkably high, almost approaching 100%. Since the introduction of ASF into China in 2018, its rapid spread has caused marked economic losses in the country’s swine industry. To date, there are no safe and effective commercial vaccines or antiviral drugs against ASF; thus, there is an urgent need to develop novel prevention and control strategies. Traditional Chinese medicine (TCM), which comprises various herbs that are abundant in various potential functional components, holds great promise for the prevention and control of ASF. Here, we provide a comprehensive review of the advancements in TCM and the effects of its compound formulas against ASF, including the antiviral abilities, immunoregulatory activities, and practical application of these formulas for the prevention and control of ASF. We specifically examined the potential and constraints of natural product-derived extracts and TCM formulas in combating ASFV. This review aims to offer insights and ideas for the holistic management and containment of ASF.
Lactobacillus species have excellent abilities to reduce intestinal inflammation and enhance gut barrier function. This study elucidated the potential mechanisms through which Lactobacillus mitigates lipopolysaccharide (LPS)-induced intestinal injury from the perspective of macrophage–intestinal epithelial cell interactions. Lactobacillus intervention improved the histopathological score; elevated ZO-1 and Occludin protein production; reduced CD16+ cell numbers; diminished IL-1β, IL-6, and TNF-α levels; decreased inducible nitric oxide synthase (iNOS) expression; increased CD163+ cell numbers; elevated IL-10 concentration; and increased arginase-1 (Arg1) expression in LPS-challenged piglets. Lactobacillus pretreatment also altered the colonic microbiota, thereby increasing the butyric acid concentration and GPR43 expression in the LPS-challenged piglets. Compared with those in the LPS group, sodium butyrate (SB) pretreatment decreased IL-1β, IL-6 and TNF-α secretion and iNOS expression but increased IL-10 secretion and Arg1 expression in macrophages. The SB-pretreated macrophages reduced the protein expression of TLR4, MyD88, and phosphorylated NF-κB p65 but increased the protein expression of ZO-1 and Occludin in intestinal epithelial cells. Moreover, GLPG0974 blocked the beneficial effects of SB on macrophages and intestinal epithelial cells. This study demonstrated that Lactobacillus improves intestinal barrier function by regulating the macrophage phenotype through the control of butyric acid and GPR43 levels to further control inflammation.
We report the development of a triplex nucleic acid lateral flow immunoassay (NALFIA) for the detection of the genomes of Nipah virus (NiV), Middle East respiratory syndrome coronavirus (MERS-CoV) and Reston ebolavirus (REBOV), which are intended for screening bats as well as other hosts and reservoirs of these three viruses. Our triplex NALFIA is a two-step assay format: the target nucleic acid in the sample is first amplified using tagged primers, and the tagged dsDNA amplicons are captured by antibodies immobilized on the NALFIA device, resulting in signal development from the binding of a streptavidin-colloidal gold conjugate to a biotin tag on the captured amplicons. Triplex amplification of the N gene of NiV, the UpE gene of MERS-CoV, and the Vp40 gene of REBOV was optimized, and three compatible combinations of hapten labels and antibodies were identified for end point detection. The lowest RNA copy numbers detected by the triplex NALFIA were 8.21e4 for the NiV N target, 7.09e1 for the MERS-CoV UpE target, and 1.83e4 for the REBOV Vp40 target. Using simulated samples, the sensitivity and specificity for MERS-CoV and REBOV targets were estimated to be 100%, while the sensitivity and specificity for the NiV target were 91% and 93.3%, respectively. The compliance rate between triplex NALFIA and real-time RT‒PCR was 92% for the NiV N target and 100% for the MERS-CoV UpE and REBOV Vp40 targets.
Mycobacterium tuberculosis (Mtb) employs multiple mechanisms, such as phagocytosis and autophagy, to evade innate immune clearance and establish infection. In the present study, we identified the ESX-1 secretion-associated protein EspL, which promotes Mtb survival by inhibiting phagosome maturation and autophagy initiation. EspL knockout decreased Mtb intracellular survival, while EspL overexpression increased bacterial survival by interfering with phagocytosis and autophagy. EspL interacts with ULK1 and promotes its phosphorylation at Ser757, leading to the inhibition of autophagy initiation. Additionally, overexpression of EspL reduced antigen presentation and T-cell responses both in vitro and in vivo. Our findings revealed that EspL interferes with autophagy and antigen presentation by suppressing ULK1 activation. These insights provide a novel understanding of Mtb pathogenicity.
Lumpy skin disease (LSD) is a highly contagious disease caused by lumpy skin disease virus (LSDV) in bovines. Rapid and accurate diagnosis is very important to controll it. However, current commercial detection kits need to be improved in terms of sensitivity or specificity. This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay (cELISA) based on the newly identified antigen gene LSDV034. The rLSDV034 protein was identified as a potential diagnostic antigen, and it was expressed, purified, and used to immunize BALB/c mice. Using laboratory-prepared indirect ELISA (iELISA), the positive cell lines were screened, and their blocking activity was further verified by competitive ELISA (cELISA). The cell line, 1H7, was chosen to produce mouse ascites, which were purified for a monoclonal antibody (mAb, 5.395 mg/mL). The heavy chain type of the 1H7 mAb was identified as IgG1a, and its light chain subtype was identified as κ. Furthermore, cELISA was developed to detect bovine serum antibodies, with rLSDV034 (4 μg/mL) as the coating antigen and HRP-1H7 mAb (1:300) as the competitive antibody. The cutoff value of cELISA was 55%, based on 32 negative bovine serum samples. The analytical sensitivity was 1:8, and no cross-reaction was detected with bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella multocida (P. multocida), or Mycoplasma bovis (M. bovis) from the serum samples. The diagnostic sensitivity and specificity of cELISA were 98.46% (95% confidence interval, CI: 91.7–100) and 100% (95% CI: 89.1–100), respectively, based on the analysis of 30 LSDV-infected bovine serum samples, 35 GTPV-vaccinated samples, and 32 negative samples. The overall coincidence of the cELISA with the virus neutralization test (VNT) reached 98.97% (95% CI: 94.4–100). Furthermore, we used cELISA to analyze 230 clinical bovine serum samples (including 59 infected and 171 vaccinated samples) and found that the serum positivity rates of the immunized samples (on d 60 postimmunization) and infected samples were 77.78% (95% CI: 70.8–83.8%) and 71.19% (95% CI: 57.9–82.2), respectively. These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.
African swine fever (ASF) is an acute and fatal hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV) that currently threatens the pig industry worldwide. Since the 2018 ASF outbreak in China, ASFV has evolved and caused diverse clinical manifestations, such as chronic and asymptomatic infections. Therefore, it is important to understand the molecular mechanisms underlying ASFV attenuation in the field. Here, we isolated three ASFVs from one diseased and two asymptomatic pigs by using primary porcine alveolar macrophages (PAMs) from both domestic pigs and Bama minipigs. The three ASFVs exhibited similar phenotypes in cell culture, including cytopathic effects (CPEs), hemadsorptions (HADs), viral protein expressions and growth curves. Genome sequencing revealed that all three ASFVs were genotype II strains. Genomic comparisons suggested that the disruption of the viral genes MGF360 and MGF110, rather than EP402R and EP153R, is likely involved in the potential attenuation of ASFV via the upregulation of innate immune responses.
The recent Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic highlights the significant threat coronaviruses (CoVs) pose to public health. With their extensive cross-continental movements, migratory birds have the potential to serve as reservoirs and vectors for CoVs. This study aimed to investigate the prevalence of CoVs in birds in densely populated areas of Guangdong Province, China. Of the 128 samples collected from birds, six tested positive for CoVs (4.7%, 95% CI: 1.7–9.9%), and three complete viral genomes were obtained through viral metagenomics and PCR. Phylogenetic analysis revealed that two CoVs (MD_XN18 and SG_DWY40) belonged to the Gammacoronavirus genus, while one (CP_XN11) belonged to the Deltacoronavirus genus. Homology analysis revealed that the MD_XN18 strain discovered in mallards shares 95.6–97.4% sequence similarity with chicken infectious bronchitis viruses (IBVs), providing direct evidence that migratory mallards can transmit avian IBVs. Recombination analysis suggested that two genomic regions of SG_DWY40 could originate from unknown sources through recombination, potentially leading to the expression of a novel viral protein, provisionally named NS3.5. These findings underscore the ongoing transmission and evolution of CoVs among birds in cities near Guangdong Province, emphasizing the need for continued monitoring and research.
1. High-fat diets result in disruption of Nyctereutes procyonoides intestinal flora.
2. High-fat diets cause functional and metabolic disturbances in the intestinal flora.
3. Disorders of gut microbiota induce inflammatory response, oxidative stress, and intestinal barrier disruption in the Nyctereutes procyonoides colon.
Single B-cell antibody generation technology is an advanced method that offers several advantages, including rapid production, high efficiency, and high yield. The antibodies generated via this technique retain their natural conformation and are well suited for applications in pathogen diagnosis, disease treatment, and investigations of virus cross-species transmission mechanisms. Our study aimed to establish a platform for generating single B-cell antibodies specifically targeting the foot-and-mouth disease virus (FMDV) 146S antigen in mice. Female BALB/c mice were immunized with inactivated O-type FMDV 146S antigen, and spleen cells were collected for further analysis. Flow cytometric sorting was performed using a biotin-labeled O-type FMDV 146S antigen as a decoy to identify and select CD19 + /CD21/35 + /CD43-/IgM-/Biotin + antigen-specific individual B cells. The gene sequences encoding the variable regions of the heavy and light chains of the murine IgG antibodies were obtained via single-cell nested PCR amplification. Separate constructs were created for the heavy and light chain plasmids to ensure the proper expression of intact IgG antibodies. These plasmids were cotransfected into human embryonic kidney 293T (HEK293T) cells, leading to the successful production and purification of 15 specific monoclonal antibodies (mAbs), 10 which exhibited activity in ELISA tests, and six antibodies that displayed activity in IFA tests. These findings highlight the successful development of a method for generating mouse-derived single B-cell antibodies that target FMDV. This achievement provides a solid foundation for diagnostic techniques and the analysis of antigenic structural variations.
Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission. It is necessary to develop an effective serological diagnostic tool for the surveillance of PDCoV infection and vaccine immunity effects. In this study, we developed a monoclonal antibody-based competitive ELISA (cELISA) that selected the purified recombinant PDCoV nucleocapsid (N) protein as the coating antigen to detect PDCoV antibodies. To evaluate the diagnostic performance of the cELISA, 122 swine serum samples (39 positive and 83 negative) were tested and the results were compared with an indirect immunofluorescence assay (IFA) as the reference method. By receiver operating characteristic (ROC) curve analysis, the optimum cutoff value of percent inhibition (PI) was determined to be 26.8%, which showed excellent diagnostic performance, with an area under the curve (AUC) of 0.9919, a diagnostic sensitivity of 97.44% and a diagnostic specificity of 96.34%. Furthermore, there was good agreement between the cELISA and virus neutralization test (VNT) for the detection of PDCoV antibodies, with a coincidence rate of 92.7%, and the κ analysis showed almost perfect agreement (κ = 0.851). Overall, the established cELISA showed good diagnostic performance, including sensitivity, specificity and repeatability, and can be used for diagnostic assistance, evaluating the response to vaccination and assessing swine herd immunity.
Trained immunity is a phenomenon in which brief exposure to an infectious agent or a vaccine can induce long-lasting changes in the host's immune system, enhancing protection against subsequent infections. The concept of trained immunity has a significant impact on the field of immunology and has the potential to revolutionize how we approach vaccination and infectious disease control. Investigations into trained immunity are rapidly advancing and have led to the development of new vaccines and immunotherapeutic strategies that harness the power of this phenomenon. While more investigations are needed to fully understand the mechanisms of trained immunity and its potential limitations, the prospects for its future application in clinical practice are promising. Here, we describe trained immunity as a biological process and explore the innate cues, epigenetic changes, and metabolic reprogramming activities that affect how trained immunity is induced.
Porcine reproductive and respiratory syndrome (PRRS) is a severe threat to pig farming worldwide and contributes to substantial financial losses. Endogenous short RNAs, known as microRNAs (miRNAs), play various roles in controlling viral infections in both human and animals through virus‒host interactions, mediating immune-related gene responses in target cells despite their unknown precise roles in infectious illnesses. Thus, a comprehensive literature search was conducted in EMBASE, PubMed and Web of Science databases to compile this review, focusing on the function, role, and involvement of miRNAs in porcine reproductive and respiratory syndrome virus (PRRSV) infection. In addition to possible exogenous miRNAs such as miR2911 and miR168, our study provides strong evidence demonstrating the important effects of endogenous miRNAs such as miR-181, miR-26, and miR-145 on PRRSV infection, which were also anticipated to bind to certain locations within PRRSV genomes. Our study highlights that miRNAs can serve as a treatment strategy of PRRS and further research is needed to validate their clinical safety and efficacy.
African swine fever (ASF) is a highly fatal hemorrhagic disease affecting domestic pigs caused by African swine fever virus (ASFV). Genetic analysis of ASFV isolates to date has identified 24 geographically related genotypes with various subgroups, but only genotype I and II ASFVs have been reported outside Africa. ASFV genotype II and genotype I viruses were reported in China in 2018 and 2021, respectively. In this study, unique and highly conserved noncoding regions were found between MGF_505-9R and MGF_505-10R in the 188 genomes of ASFV genotypes I and II. A pair of primers was designed on the basis of this region. By optimizing the reaction system and conditions, a SYBR Green I fluorescence PCR assay that can distinguish between ASFV genotypes I and II was established, and the sensitivity, reproducibility and specificity were evaluated. The detection limit was 1 TCID50/0.1 mL for both genotypes, with no cross-reactivity observed with other common pig pathogens. The intra- and interbatch variation coefficients were both less than 1.2%. Clinical sample detection analysis revealed 47 positive cases out of 100, including 3 for genotype I and 44 for genotype II, aligning with results from the WOAH-recommended and national standard methods. The method developed in this study allows for the differentiation of ASFV genotypes I and II without the need for genome sequencing, offering a convenient and rapid approach for ASFV detection and genotype identification.
Oxidative stress is an important contributor to gastrointestinal diseases in multiple ways. Taurodeoxycholic acid (TDCA) is a metabolite of bile acids and has anti-inflammatory and protective effects on the intestinal tract. However, whether TDCA can alleviate oxidative stress in the intestine is still unclear. Here, we investigated the effects of TDCA on diquat-induced oxidative stress in the jejunum and its mechanism. The results revealed that TDCA increased the concentrations of antioxidant enzymes in the serum, jejunal tissue and intestinal epithelial cells of the mice, as did the expression of tight junction-associated proteins and the Nrf2 protein in the jejunal epithelial tissue and intestinal epithelial cells. We then explored the mechanism of Nrf2 with ML385 (a specific Nrf2 inhibitor). The results showed that after ML385 treatment, the levels of antioxidant enzymes were significantly decreased in the serum, jejunum, and intestinal epithelial tissues of the mice. The expression of tight junction proteins in jejunum epithelial tissues and intestinal epithelial cells was also decreased. In conclusion, our study suggests that TDCA alleviates oxidative stress to improve intestinal barrier function through the Nrf2-mediated signaling pathway. These findings help elucidate the role of TDCA in protecting the intestinal barrier and its mechanism of action, providing insights for the prevention and treatment of intestinal diseases caused by oxidative stress.
Emerging infectious diseases are a major threat to biodiversity and an important public health issue. Flaviviruses are the cause of several emerging vector-borne zoonotic arboviruses whose distribution is currently increasing in Europe. The evidence that West Nile virus (WNV) circulates in resident and migratory species has implications for both animal and public health and should therefore be studied in depth. USUTU (USUV), Bagaza (BAGV) and tick-borne encephalitis virus (TBEV) are other viruses that are beginning to spread more widely. An integrated surveillance program, namely in birds, is essential for reducing the risk of infection in human populations within the One Health principles. In the present study, wild birds admitted to wildlife rehabilitation centers in Portugal were sampled. Two hundred eight blood samples were assayed serologically for antibodies to flaviviruses by using a commercial ELISA kit. An overall seroprevalence of 19.6% (95% confidence interval [CI]: 13.7–26.7%) was observed. Antibodies against flaviviruses were detected in 13 (35.1%) different species of wild birds. Accipitriformes (26.7%; 95% CI: 18.5–36.2%) and Strigiformes (26.7%; 95% CI: 14.6–42.0%) were the orders with the highest seroprevalence rates recorded. There were no statistically significant differences (p = 0.725) between the geographical regions (NUTS II) studied, but a statistically significant difference (p = 0.017) was found between sex (male: 34.4%; female: 4.8%). A higher seroprevalence was detected in adults (32.1%) than in juvenile birds (9.3%) (p = 0.014), and age was considered a risk factor for flavivirus infection in wild birds (odds ratio 1.4; 95% CI: 0.5–4.0). More epidemiological studies are needed in Portugal since the actual spread of the genus Flavivirus throughout the country is unknown.
Pathogenic African swine fever virus (ASFV) remains a lethal causative agent in the domestic pig industry, which poses a burden on the swine market and causes substantial socioeconomic losses worldwide. Currently, there are no commercially efficacious vaccines or specific treatments available for ASF prevention and control. Unfortunately, little is known about the swine immune response upon ASFV infection. Here, we investigated the host immune response discrepancy induced by the field moderately virulent strain ASFV HB-2208 among healthy, diseased and asymptomatic pigs. In the peripheral blood of diseased swine, lymphopenia is caused by the massive loss of bystander lymphocytes, such as γδ T cells, B cells and CD4+ T cells. Conversely, ASFV has a strong tropism for the mononuclear phagocyte system (MPS) and partial dendritic cells (DCs), whose antigen-presenting ability is impeded by the downregulation of CD80 and MHC I. However, no significant difference in the number of CD8αhigh T cells was detected, whereas the frequencies of NK cells, NKT cells, and regulatory T cells (Tregs) were significantly increased. Additionally, an in vitro model was established with a coculture of primary pulmonary alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs), which significantly reduced γδ T cells, B cells and CD4+ T cells and increased Tregs. The differentiated immune response might aid in enhancing the understanding of ASFV pathogenesis in suids and provide insights into the mechanism of ASFV-induced lymphopenia for further studies.