The primary aim of this study was to evaluate the efficacy of phage against mastitis induced by drug-resistant S. aureus in a mouse model. In this study, five S. aureus phages—4086-1, 4086-2, 4086-3, 4086-4, and 4086-6—were isolated from milk samples secreted by mastitis cows. Transmission electron microscopy showed that all the five phages had icosahedral heads and short non-contractile tails, which are typical characteristics of the family Podoviridae. All these phages were species-specific against S. aureus. The one-step growth curve showed a short latency period (10–20 min) and high burst size (up to 400 PFU/infected cell). To evaluate the effectiveness of the phage 4086-1 in the treatment against mastitis, a mouse model of mastitis was challenged with drug-resistant S. aureus. The results showed the proliferation of S. aureus in the mammary glands was significantly inhibited after treating by phage 4086-1. The concentrations of TNF-α and IL-6 decreased significantly, which demonstrated the phages could effectively alleviate the inflammatory responses. Furthermore, the histopathological analysis showed that inflammatory infiltration in the mammary glands was significantly reduced. These results demonstrate that phage may be a promising alternative therapy against mastitis caused by drug-resistant S. aureus.

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. Key points: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Porcine epidemic diarrhea virus (PEDV) is a rapidly evolving virus that causes outbreaks in pig herds worldwide. Mutations in the S protein of PEDV have led to the emergence of new viral variants, which can reduce vaccine immunity against prevalent strains. To understand the infection and variation pattern of PEDV in China, an extensive epidemiological survey was conducted in northeast China from 2015 to 2022. The genetic diversity of enteroviruses co-infected with PEDV and the PEDV S gene was analyzed, common mutation patterns that may have led to changes in PEDV virulence and infectivity in recent years were identified, and structural changes in the surface of the S protein resulting from mutations in the PEDV S gene from 2011 to 2022 were reviewed. Of note, two distinct mutations in the emerging 2022 HEB strain were identified. These findings provide a basis for a better understanding of PEDV co-infection and genetic evolution in northeast China.

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (alr), in the genome of Lactobacillus casei strain W56 (L. casei W56) was knocked out to construct an auxotrophic L. casei strain (L. casei Δalr W56) using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/Δalr W56) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection.

Autophagy is an important cellular response against intracellular pathogens. However, some viruses have evolved mechanisms to hijack this defensive process to provide favorable conditions for virus replication in host cells. The porcine epidemic diarrhea virus (PEDV) has been shown to alter autophagy pathways; however, it is still unknown through which receptors PEDV induces autophagy in IPEC-J2 cells, whether autophagy facilitates PEDV replication, and which functional domains of PEDV proteins are primarily responsible for inducing autophagy. Here, we found that PEDV infection induces autophagy in host cells via distinct and uncoupled molecular pathways. RNA-seq technology was used to analyze the expression patterns of intracellular genes in PEDV-infected IPEC-J2 cells using transcriptomics. The results demonstrate that PEDV triggers autophagy via the cellular pathogen receptor TLR4 and the AKT-mTOR pathway. As evidenced by autophagosome detection, PEDV infection increases autophagosomes and light chain 3 (LC3)-II as well as downregulated AKT-mTOR phosphorylation. Our study revealed that the binding of the viral protein NSP61-2C (56-151aa) to TLR4 triggers autophagy and inactivates the AKT-mTOR pathway, both of which are critical for facilitating PEDV infection. Through screening and analysis, TLR4 was found to be a key gene involved in PEDV-induced autophagy. The screening of the key functional domains of NSP6 (56-151aa) for their ability to induce autophagy in IPEC-J2 cells provided a basis for the in-depth analysis of the pathogenic mechanism of PEDV infection-induced autophagy and promotion of self-replication and also provided an important target for the study of PEDV antiviral drugs. In conclusion, we elucidated that the PEDV infection of IPEC-J2 cells could induce autophagy and found that PEDV could use autophagy to promote its own replication.