Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Cloning, expression, and characterization of thermotolerant manganese superoxide dismutase from Bacillus sp. MHS47.

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

The efficacy of a single-oral-dose administration of ivermectin and diethylcarbamazine on the treatment of feline Brugia malayi.

The combination of ivermectin and diethylcarbamazine (DEC) have been shown to be superior to either drug alone for the suppression of Brugia malayi in humans, but their efficacy against infection with B. malayi in cats has never been investigated. Fourteen asymptomatic microfilaremic (1-200 microfilariae/20 microl blood) cats received oral doses of ivermectin (400 microg/kg body weight) and DEC (6 mg/kg body weight) as a single treatment. A two-month post-treatment examination revealed that 87-100% of the microfilariae in each subject had been cleared, with two of the subjects being amicrofilaremic. A further reduction in microfilarial levels was observed until the final follow-up, at 8 months post-treatment, when the mean clearance rate was 99% and 12 out of the 14 subjects (86%) were amicrofilaremic. The combination of ivermectin and DEC demonstrated a microfilaricidal effect superior to that of either drug used alone, both in the initial rapid clearance of microfilariae, and in sustaining the effect for 8 months. This finding has important implications for the control of brugian lymphatic filariasis in the cat reservoir.