RPA-CRISPR/Cas12a-Based Detection of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/µL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level.

Accelerated loop-mediated isothermal amplification method for the rapid detection of Streptococcus suis serotypes 2 and 14 based on single nucleotide polymorphisms.

Streptococcus suis serotypes 2 and 14 are the most prevalent zoonotic strains. The establishment of a sensitive and extremely accurate method for point-of-care testing for Streptococcus suis serotype 2 and 14 strains is highly desirable. In this study, a loop primer probe-introduced loop-mediated isothermal amplification assay was developed to differentiate Streptococcus suis serotypes 2 and 14 based on SNP (single nucleotide polymorphism). The specific fluorescent probes were designed for the SNP site specific for serotype 2 and 14 Streptococcus suis cpsK genes, and the loop primer probe-introduced loop-mediated isothermal amplification (LAMP) assay was developed using the specific cleavage properties of the RNase H2 enzyme. Rapid and efficient LAMP assays were realized through the use of loop forward primers and stem forward primers. The results showed that the amplification reaction can be performed efficiently at 59°C. The results can be real-time detected or judged using a smartphone and a 3D-printed visualization cassette. The sensitivity of the LAMP assay can reach 18.4 CFU within 40 minutes. The detection rate of the assay system was evaluated using 19 clinical samples with suspected Streptococcus suis infection, and the detection rate was consistent with the sequencing method, suggesting that the test is highly practical. The LAMP assay for Streptococcus suis serotypes 2 and 14 established in this study has strong specificity, high sensitivity, and simple operation, while the reaction can be performed at an isothermal temperature and is not dependent on complex instruments or professional operators, making it suitable for field testing.

Proteome Analysis of Outer Membrane Vesicles From a Highly Virulent Strain of Haemophilus parasuis.

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3.

Since 2016, a novel porcine circovirus, PCV3, has been infecting pigs, causing significant economic losses to the pig industry. In recent years, the infection rate of PCV3 has been increasing, and thus rapid and accurate detection methods for PCV3 are essential. In this study, we established a novel probe-based single multiple cross displacement amplification (P-S-MCDA) method for PCV3. The method was termed as P-S-MCDA. The P-S-MCDA uses seven primers to amplify the capsid gene, and the assay can be performed at 60°C for 30 min, greatly shortening the reaction time. The results of P-S-MCDA can not only be monitored in real time through fluorescence signals but also be determined by observing the fluorescence of the reaction tubes using a smartphone-based cassette. This method demonstrated good specificity and the same sensitivity as qPCR, with a minimum detection limit of 10 copies. In 139 clinical samples, the coincidence rate with qPCR was 100%. The P-S-MCDA can be widely applied in PCV3 detection in laboratories or in rural areas.

RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells.

Pigs infected by pseudorabies virus (PRV) display necrotic pathology in multiple organs. The mechanism by which PRV induces cell death is still unclear. Recently, necroptosis was identified as a programmed process dependent on the receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase-like protein (MLKL). In this study, we demonstrated that PRV induced RIPK3-dependent necroptosis in PK-15 cells. The data showed that PRV infection caused cell death with Propidium Iodide (PI)-positive staining. Transmission electron microscopy analysis indicated plasma membrane disruption in PRV-infected cells. A pan-caspase inhibitor did not prevent PRV-induced necrotic cell death. Western blot analysis indicated that caspase-3 and caspase-8 were not cleaved during PRV infection. Although the transcription of tumor necrosis factor-alpha (TNF-α) was increased by PRV infection, RIPK1 was shown to be not involved in PRV-induced necrotic cell death by use of its specific inhibitor. Further experiments indicated that the phosphorylation of RIPK3 and MLKL was upregulated in PRV-infected cells. Stable shRNA knockdown of RIPK3 or MLKL had a recovery effect on PRV-induced necrotic cell death. Meanwhile, viral titers were enhanced in RIPK3 and MLKL knockdown cells. Hence, we concluded that initiation of necroptosis in host cells plays a limiting role in PRV infection. Considering that necroptosis is an inflammatory form of programmed cell death, our data may be beneficial for understanding the necrotic pathology of pigs infected by PRV.

Comparison of Host Cytokine Response in Piglets Infected With Toxigenic and Non-toxigenic Staphylococcus hyicus.

Staphylococcus hyicus is the most common causative agent of exudative epidermitis (EE) in piglets. Staphylococcus hyicus can be grouped into toxigenic and non-toxigenic strains based on its ability to cause EE in pigs. However, the inflammatory response of piglets infected with toxigenic and non-toxigenic S. hyicus has not been elucidated. In this study, we evaluated the serum cytokine profile in piglets inoculated with toxigenic and non-toxigenic S. hyicus strains and recorded the clinical signs in piglets. Fifteen piglets were divided into three groups (n = 5) and inoculated with a toxigenic strain (ZC-4), a non-toxigenic strain (CF-1), and PBS (control), respectively. The changes in serum levels of cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, interferon-γ, transforming growth factor-ß1, and tumor necrosis factor-α) were evaluated using a cytokine array at 6, 24, 48, and 72 h post inoculation. The results showed that piglets infected with the toxigenic strain exhibited more severe clinical signs and higher mortality than those infected with the non-toxigenic strain. The serum levels of pro-inflammatory cytokine IL-1ß were significantly increased in toxigenic-and non-toxigenic-strain-infected piglets compared to those in the control group (p < 0.05), while the anti-inflammatory cytokine IL-10 was significantly up-regulated only in toxigenic group than in control group (p < 0.05). These results indicated that piglets infected with toxigenic and non-toxigenic S. hyicus showed differential infection status and inflammatory responses. Both toxigenic- and non-toxigenic- S. hyicus infection could induce a pro-inflammatory reaction in piglets. In addition, the toxigenic strain induced a strong anti-inflammatory response in piglets as indicated by the increased serum level of IL-10, which may be associated with the severe clinical signs and increased mortality and may be the key cytokine response responsible for pathogenic mechanisms of S. hyicus.

The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3.

In 2016, a novel porcine circovirus (PCV), PCV3, was identified in USA. Subsequently, it was proved to be also epidemic in China, Poland, and Korea. To analyze and control the epidemic situation of PCV3, it is necessary to establish accurate and high-throughput detection methods. In this study, the colorimetric isothermal multiple-self-matching-initiated amplification (IMSA) using cresol red was developed to detect PCV3 for the first time. The reaction can be easily performed by incubating the tube at 63°C for 60 min. By the addition of pH-sensitive indicator dye cresol red, the initial color of the reaction mixture is red. When PCV3 capsid gene DNA was positive in the sample, the color of the reaction mixture changed from red to yellow after the isothermal incubation at 63°C, while the negative control maintained the red color. The colorimetric IMSA displayed good specificity in detecting PCV3, PCV2, and PCV1 and 4 porcine DNA pathogens. Moreover, it has a low and repeatable detection limit of 10 copies, which is consistent with TaqMan-based qPCR, but 10 times more sensitive than PCR. In diagnosing 128 clinical specimens, it not only showed 100% agreement with qPCR but also detected 15 positive results more than PCR. The colorimetric IMSA we offered might be a good choice for PCV3 epidemiological investigation and point-of-care testing.

Metabolomics Exploration of Pseudorabies Virus Reprogramming Metabolic Profiles of PK-15 Cells to Enhance Viral Replication.

For viral replication to occur in host cells, low-molecular-weight metabolites are necessary for virion assembly. Recently, metabolomics has shown great promise in uncovering the highly complex mechanisms associated with virus-host interactions. In this study, the metabolic networks in PK-15 cells infected with a variant virulent or classical attenuated pseudorabies virus (PRV) strains were explored using gas chromatography-mass spectrometry (GC-MS) analysis. Although total numbers of metabolites whose levels were altered by infection with the variant virulent strain or the classical attenuated strain were different at 8 and 16 h post infection (hpi), the predicted levels of differential metabolic components were shown to be associated with specific pathways, including glycolysis as well as amino acid and nucleotide metabolism. The glucose depletion and glycolysis inhibitors 2DG and oxamate could reduce the level of PRV replication in PK-15 cells. In addition, the inhibition of the pentose phosphate pathway (PPP) resulted in an obvious decline of viral titers, but the prevention of oxidative phosphorylation in the tricarboxylic acid (TCA) cycle had a minimal effect on viral replication. Glutamine starvation resulted in the decline of viral titers, which could be restored by supplemental addition in the culture media. However, inhibition of glutaminase (GLS) activity or the supplement of 2-ketoglutarate into glutamine-deleted DMEM did not alter PRV replication in PK-15 cells. The results of the current study indicate that PRV reprograms the metabolic activities of PK-15 cells. The metabolic flux from glycolysis, PPP and glutamine metabolism to nucleotide biosynthesis was essential for PRV to enhance its replication. This study will help to identify the biochemical materials utilized by PRV replication in host cells, and this knowledge can aid in developing new antiviral strategies.