Phages in sludge from the A/O wastewater treatment process play an important role in the transmission of ARGs.

Phages can influence the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) through transduction, but their profiles and effects on the transmission of ARGs are unclear, especially in complex swine sludge. In this study, we investigated the characterization of phage and ARG profiles in sludge generated from anoxic/oxic (A/O) wastewater treatment processes on swine farms using metagenomes and viromes. The results demonstrated that 205-221 subtypes of ARGs could be identified in swine sludge, among which sul1, tet(M), and floR were the dominant ARGs, indicating that sludge is an important reservoir of ARGs, especially in sludge (S) tanks. The greater abundance of mobile genetic elements (MGEs) in the S tank could significantly contribute to the greater abundance of ARGs there compared to the anoxic (A) and oxic (O) tanks (P < 0.05). However, when we compared the abundances of ARGs and MGEs in the A and O tanks, we observed opposite significant differences (P < 0.05), suggesting that MGEs are not the only factor influencing the abundance of ARGs. The high proportion of lysogenic phages in sludge from the S tank can also have a major impact on the ARG profile. Siphoviridae, Myoviridae, and Podoviridae were the dominant phage families in sludge, and a network diagram of bacteria-ARG-phages revealed that dominant phages and bacteria acted simultaneously as potential hosts for ARGs, which may have led to phage-mediated HGT of ARGs. Therefore, the risk of phage-mediated HGT of ARGs cannot be overlooked.

Diltiazem HCl suppresses porcine reproductive and respiratory syndrome virus infection in susceptible cells and in swine.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen for swine, resulting in substantial economic losses to the swine industry. However, there has been little success in developing effective vaccines or drugs for PRRSV control. In the present study, we discovered that Diltiazem HCl, an inhibitor of L-type Ca2+ channel, effectively suppresses PRRSV replication in MARC-145, PK-15CD163 and PAM cells in dose-dependent manner. Furthermore, it demonstrates a broad-spectrum activity against both PRRSV-1 and PRRSV-2 strains. Additionally, we explored the underlying mechanisms and found that Diltiazem HCl -induced inhibition of PRRSV associated with regulation of calcium ion homeostasis in susceptible cells. Moreover, we evaluated the antiviral effects of Diltiazem HCl in PRRSV-challenged piglets, assessing rectal temperature, viremia, and gross and microscopic lung lesions. Our results indicate that Diltiazem HCl treatment alleviates PRRSV-induced rectal temperature spikes, pulmonary pathological changes, and serum viral load. In conclusion, our data suggest that Diltiazem HCl could serve as a novel therapeutic drug against PRRSV infection.

Glycerol Monolaurate to Ameliorate Efficacy of Inactivated Pseudorabies Vaccine.

The present study is aimed to evaluate the effect of glycerol monolaurate (GML) on the growth performance and immune enhancement of pseudorabies virus (PRV)-inactivated vaccine in the early-weaned piglets. One hundred and twenty-five 28-day-old weaned piglets were randomly assigned to a control group (CON, no vaccine and no challenge), challenge control group (C-CON), inactivated PRV vaccine group (IPV), IPV + 500 mg/kg GML group (L-GML), and IPV + 1,000 mg/kg GML group (H-GML) during the entire 28-day experimental period. All the data analyses were performed by one-way analysis of variance (ANOVA) and multiple comparisons. Our results showed that the final weight, average daily gain (ADG), and average daily feed intake (ADFI) of H-GML were the highest in each group, and F/G of H-GML was increased but there was no significant difference with CON (p > 0.05). Levels of PRV glycoprotein B (gB) antibody and immunoglobulin in serum of L-GML and H-GML were higher than those of IPV, but only gB antibody levels and immunoglobulin G (IgG) in H-GML were significantly increased (p < 0.05). Compared with IPV, the contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in serum of L-GML (TNF-α and IL-1ß: p > 0.05, IL-6: p < 0.05, respectively) and H-GML (p < 0.01, both) were all decreased, and the content of interleukin-10 (IL-10) in H-GML was increased (p > 0.05). Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) experiments proved that L-GML and H-GML were both superior to IPV in inhibiting the expression of TNF-α (p < 0.01), IL-6 (p > 0.05), and IL-1ß (p < 0.01) mRNAs and promoting the expression of IL-10 mRNA (L-GML: p > 0.05, H-GML: p < 0.05, respectively) in the superficial inguinal lymph nodes. Histopathological examination found mild congestion in the lung and inguinal lymph nodes of IPV, while the tissues (brain, lung, and inguinal lymph nodes) of L-GML and H-GML were the same as CON with no obvious lesions. The above results indicate that GML may improve the growth performance of weaned piglets and enhance the immunity of PRV-inactivated vaccine by increasing the levels of PRV gB antibody and immunoglobulin and regulating cytokine levels.

Same Dosages of rPRV/XJ5-gI-/gE-/TK- Prototype Vaccine or Bartha-K61 Vaccine Similarly Protects Growing Pigs Against Lethal Challenge of Emerging vPRV/XJ-5 Strain.

Variant pseudorabies viruses (vPRV) have constantly emerged in China since late 2011. In the present study, a 1 × 106.0 TCID50 per-animal dosage of a commercially available Bartha-K61 vaccine and an rPRV/XJ5-gI-/gE-/TK- prototype vaccine freshly extracted from the vPRV/XJ-5 at the same dose were administered to evaluate the immune effectiveness thereof on growing pigs to prevent lethal strikes caused by vPRV/XJ-5. The results suggest that the Bartha-K61 vaccine at a dose of 1 × 106.0 TCID50 per animal and the same dosage of the rPRV/XJ5-gI-/gE-/TK- prototype vaccine protected growing pigs against the lethal challenge of vPRV/XJ-5 strain with 100% survive rate. Furthermore, the outcome of the clinical score, virus shedding, weight gain, and viral loads in different pig tissues in these two groups demonstrates that either the Bartha-K61 vaccine or the rPRV/XJ5-gI-/gE-/TK- prototype vaccine at the same dose exhibited parallel efficacy in pigs against the lethal challenge with the XJ-5 strain of vPRV.

The Epidemiological Analysis of Pseudorabies Virus and Pathogenicity of the Variant Strain in Shandong Province.

Pseudorabies (PR) is a disease that is seriously endangering the pig industry in China. To understand the current prevalence of pseudorabies virus (PRV) in Shandong Province, China, 19,292 serum samples were collected from 16 locations in Shandong from 2018 to 2020. The gE antibody was detected by enzyme-linked immunosorbent assay. Ninety-seven suspected cases of PRV infection were collected from sick pigs vaccinated with Bartha-K61 to isolate PRV. The results showed that the average positive rate of the PRV gE antibody decreased from 38.20% in 2018 to 18.12% in 2020, but there was a high positive rate in sows. The isolation rate of PRV was 13.40% (13/97), and four strains were purified through plaque assay (named PRV-SD1, PRV-SD2, PRV-SD3, and PRV-SD4). The homology and genetic evolution of four PRV strains based on gE, gC, gI, and TK genes were analyzed and showed that these four strains shared more than 99.0% nucleotide homology with the variant PRV XJ5 strain, and they clustered in the same sub-branch with the domestic variant PRV strains, including JS-2012 and XJ5. Furthermore, the pathogenicity of the isolated variant strain was assessed by intranasal infection of 16-week-old pigs with 1 mL PRV-SD1 strain. The results of the animal experiment demonstrated that the PRV-SD1-infected pigs exhibited obvious clinical symptoms as early as 2 days post inoculation (dpi), and all infected pigs died within 1 week. The severe hyperemia of meninges and swelling of lungs and tonsils were observed. Histopathology analysis showed the obvious lymphocytes necrosis of tonsils, interstitial pneumonia, and viral encephalitis. Many positive staining cells were observed in tonsils and brains through immunohistochemistry staining assay. Viral shedding in oropharyngeal and rectal swabs were detected at 2 dpi, reached a peak at 3 dpi, and then gradually decreased. The detection of viral loads in the tissues showed that tonsils had the highest virus titer, further proving it may be the target organ of variant PRV infection. In conclusion, variant PRV strains were still highly prevalent in Shandong Province, and they had a strong pathogenicity in pigs.