Blocking pathogenic Leptospira invasion with aptamer molecules targeting outer membrane LipL32 protein.

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).

The Rapid Detection of Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus via Polymerase Chain Reaction Combined with Magnetic Beads and Capillary Electrophoresis.

Food safety concerns regarding foodborne pathogen contamination have gained global attention due to its significant implications. In this study, we developed a detection system utilizing a PCR array combined with an automated magnetic bead-based system and CE technology to enable the detection of three foodborne pathogens, namely Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. The results showed that our developed method could detect these pathogens at concentrations as low as 7.3 × 101, 6.7 × 102, and 6.9 × 102 cfu/mL, respectively, in the broth samples. In chicken samples, the limit of detection for these pathogens was 3.1 × 104, 3.5 × 103, and 3.9 × 102 cfu/g, respectively. The detection of these pathogens was accomplished without the necessity for sample enrichment, and the entire protocols, from sample preparation to amplicon analysis, were completed in approximately 3.5 h. Regarding the impact of the extraction method on detection capability, our study observed that an automated DNA extraction system based on the magnetic bead method demonstrated a 10-fold improvement or, at the very least, yielded similar results compared to the column-based method. These findings demonstrated that our developed model is effective in detecting low levels of these pathogens in the samples analyzed in this study. The PCR-CE method developed in this study may help monitor food safety in the future. It may also be extended to identify other foodborne pathogens across a wide range of food samples.

Combining Direct PCR Technology and Capillary Electrophoresis for an Easy-to-Operate and Highly Sensitive Infectious Disease Detection System for Shrimp.

Infectious diseases are considered the greatest threat to the modern high-density shrimp aquaculture industry. Specificity, rapidity, and sensitivity of molecular diagnostic methods for the detection of asymptomatic infected shrimp allows preventive measures to be taken before disease outbreaks. Routine molecular detection of pathogens in infected shrimp can be made easier with the use of a direct polymerase chain reaction (PCR). In this study, four direct PCR reagent brands were tested, and results showed that the detection signal of direct PCR in hepatopancreatic tissue was more severely affected. In addition, portable capillary electrophoresis was applied to improve sensitivity and specificity, resulting in a pathogen detection limit of 25 copies/PCR-reaction. Juvenile shrimp from five different aquaculture ponds were tested for white spot syndrome virus infection, and the results were consistent with the Organization for Animal Health's certified standard method. Furthermore, this methodology could be used to examine single post larvae shrimp. The overall detection time was reduced by more than 58.2%. Therefore, the combination of direct PCR and capillary electrophoresis for on-site examination is valuable and has potential as a suitable tool for diagnostic, epidemiological, and pathological studies of shrimp aquaculture.

Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes.

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.

Replication of a Dog-Origin H6N1 Influenza Virus in Cell Culture and Mice.

The world's first natural avian-origin H6N1 influenza A virus infection case in dogs was confirmed in Taiwan in 2014. The H6N1 virus in chickens has been endemic in Taiwan since 1972. Whether the dog H6N1 virus has interspecies transmission potential is the key issue we aim to understand. Following one virus passage in embryonated eggs and two further passages in MDCK cells, we obtained two virus derivatives, E01EE (PB1 739E and PB2 627E) and E01GK (PB1 739G and PB2 627K), respectively. The pathogenicity of E01EE and E01GK was investigated using plaque assay, growth dynamic analysis and cell viability quantification in cells from different animal species. The impact of amino acid mutation on PB1 739 and PB2 627 on viral ribonucleoprotein (RNP) activity was also analyzed. Further mouse infection experiments were performed. The results showed that both E01EE and E01GK decreased cell relative viability of canine MDCK cells, human A549 cells and chicken DF1 cells. E01Gk caused greater cellular harm in MDCK and A549 cells and had significantly higher virus titers in all of the cells compared to E01EE. The PB2 627K but not PB1 739G was the critical mutation that influenced the viral RNP activity. Both E01EE and E01GK caused mice pneumonia and considerable virus shedding, especially E01GK. This report verifies PB2 E627K mutation in virulence and spotlights the potential for the dog H6N1 virus to extend interspecies transmission.

Reborn from the Ashes: Turning Organic Molecules to Antimicrobial Carbon Quantum Dots.

Using polyamines as the initial organic raw material and by applying simple pyrolysis methods, super cationic carbon quantum dots (CQDs) can easily be made. Since polyamines are natural products and the synthesis procedure is green, these polyamine-derived CQDs display low toxicity and high biocompatibility but possess high antibacterial activity. In addition, polyamine-derived CQDs display other unique properties, such as facilitation of wound healing and passage through the tight junction, which make them a very promising bactericide in future clinical applications.