Hyeng-il Cheun

Detection of anthrax spores from the air by real-time PCR

Aims: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure.

Effect of the Lower Molecular Capsule Released from the Cell Surface of Bacillus anthracis on the Pathogenesis of Anthrax

Bacillus anthracis enters the body as an endospore, and encapsulation and toxin production occur after germination. Capsule is proposed to be an antiphagocytic factor, and toxin induces cytokine production for systemic shock. The dep gene, adjacent to the cap region for the encapsulation, degrades the high-molecular weight capsule (H-capsule) to the lower-molecular weight capsule (L-capsule), which releases into the culture supernatant. This study analyzed the biological function of the cap-dep region. The dep null mutant Sm-1, which formed H-capsule but not L-capsule, was avirulent. However, Sm-1 with an intact dep gene or with purified L-capsule recovered its pathogenicity. Sm-1 was subjected to phagocytosis by macrophages more easily than its parent strain, Sm, in vitro; in vivo, it cleared without L-capsule and grew well with L-capsule, which suggests that L-capsule is essential for in vivo multiplication. Moreover, a new name, capD, might be appropriate, because of the part of the cap operon involved in both polymerization and depolymerization of the capsule.

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.

Protective immunity of SpaA‐antigen producing Lactococcus lactis against Erysipelothrix rhusiopathiae infection

Aims:  To develop an economical, safe and simple vaccination system against swine erysipelas using SpaA‐antigen producing Lactococcus lactis.

A Simple and Sensitive Detection System for Bacillus anthracis in Meat and Tissue

Aims: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures.

Rapid and effective detection of anthrax spores in soil by PCR

Aims: To detect Bacillus anthracis DNA from soil using rapid and simple procedures.

The First Outbreak of Giardiasis with Drinking Water in Korea

Objectives: To identify the pathogen of the diarrhea outbreak in a village in Jeollabuk province in Korea in April 2010. Methods: DNA extraction was performed from the 120 L of collected water, which was centrifuged at 10,000 x g for 30 min. PCR reactions were conducted in a total of 25 ul, which included PCR premix (GenDEPOT, Barker, TX, USA), 2 ul (∼100 ng) of extracted DNA, and 10 pmol of each primer. Results: Nine people out of 25 had a symptom of abdominal pain accompanied by diarrhea after they used stored valley water in a water tank as a provisional water supply source without chlorine sterilization. Among them Giardia lamblia was detected in fecal samples of 7 people using the polymerase chain reaction method. Although G. lamblia was also detected from water provided by the provisional water supply system stored in the water tank and used as drinking water, it was not detected in the water tank itself. This water-borne outbreak is considered to have occurred when the provisional water supply tube was destroyed under a building construction and contaminated by G. lamblia, but its precise cause has not been clarified. Conclusion: This outbreak resulting from G. lamblia is very meaningful as the first outbreak of an infection by a water-borne parasite in Korea.

PCR Diagnosis of Entamoeba histolytica Cysts in Stool Samples

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.