Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.