Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity.

Detection of Encephalitozoon spp. from human diarrheal stool and farm soil samples in Korea.

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.

Identification of a picornavirus from chickens with transmissible viral proventriculitis using metagenomic analysis.

Transmissible viral proventriculitis (TVP), an infectious disease in chickens, is responsible for economic losses in the commercial poultry industry. The major etiologic agent, however, is unknown. Using metagenomics, we compared the diversity of viruses present in proventriculus samples from flocks diagnosed with TVP to those of healthy flocks in South Korea between 2003 and 2012. Each sample had a mean of 21,538,726 sequence reads generated by high-throughput sequencing, with a mean length of 160 nt. Enrichment in viral sequences suggested that at least three viruses were present in each TVP sample. Although we could not determine a pathogen of TVP that matched the known morphology, picornavirus sequences were present in all five disease samples, suggesting an association with TVP. The five samples yielded 1,045-1,720 bp contigs with 81-84 % nt sequence identity to turkey hepatitis virus (accession number: HM751199). Whole-genome analysis indicated that the QIA01 strain of the novel picornavirus was similar to turkey hepatitis virus in the P2 and P3 regions (82.7 % nt and 95.5 % aa sequence identity), but different in the structural region and partial 2A peptides (56.2 % nt and 23.9 % aa sequence identity). In addition, the QIA01 virus was similar (87.0 % nt and 95.6 % aa sequence identity) to chicken megrivirus, recently detected in chickens with malabsorption syndrome in Hungary. Our results are useful for understanding the genetic diversity of avian picornaviruses and for classifying chicken megrivirus as a pathogen affecting the digestive tract of chickens.

Detection of Cryptosporidium parvum in environmental soil and vegetables.

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidial enteritis. Numerous outbreaks of cryptosporidiosis have been reported worldwide. Cryptosporidium is transmitted to hosts via consumption of contaminated water and food but also by direct contact with contaminated soil or infected hosts. The present study investigated farm soil collected from 34 locations along the western Korean peninsula and 24 vegetables purchased from local grocery markets in Seoul. The soil and vegetable samples were examined by real-time polymerase chain reaction (qPCR) to estimate the risk of infection. Eleven of 34 locations (32.4%) and 3 of 24 vegetable samples (12.5%) were contaminated with Cryptosporidium parvum, as confirmed by TaqI enzyme digestion of qPCR products and DNA sequencing. It is suggested that Cryptosporidium infection can be mediated via farm soil and vegetables. Therefore, it is necessary to reduce contamination of this organism in view of public health.

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.

Recombinant thioredoxin peroxidase from Cryptosporidium parvum has more powerful antioxidant activity than that from Cryptosporidium muris.

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-irradiation. In an animal study, more than 25kGy of γ-irradiation was necessary to eliminate C. parvum infectivity from mice. In contrast, Cryptosporidium muris (murine Cryptosporidium), which lives in stomach epithelium, lost its infectivity in mice with 1kGy of γ-irradiation. Recently, it was found that thioredoxin peroxidase was highly expressed in C. parvum oocysts irradiated with high doses of γ-irradiation. Therefore we hypothesize that antioxidant activity of the thioredoxin peroxidase is involved in the radioresistance of C. parvum. To verify this, thioredoxin peroxidases of C. parvum (CpTPx) and C. muris (CmTPx) were expressed in Escherichia coli cells, and their antioxidant activities were compared. Both CpTPx and CmTPx belong to the 2-Cys family of peroxiredoxins. Hydrogen peroxide consumption was approximately 2- to 12-fold greater in recombinant CpTPx (rCpTPx) than in recombinant CmTPx (rCmTPx) in the presence of 0.2mM dithioerythritol or glutathione (GSH), respectively. The peroxidase activity of rCpTPx was highly enhanced by GSH, but that of rCmTPx was not. The minimum dose of rCpTPx required to protect supercoiled plasmid DNA from damage by metal-catalyzed oxidation was only 12% of that required with rCmTPx. The results showed that rCpTPx has more powerful antioxidant activity than rCmTPx. Further investigations on the role of CpTPx in the radioresistance of C. parvum are warranted.

Characterization of the thioredoxin peroxidase from Cryptosporidium parvum.

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-radiation. Recently, it was found that the expression of thioredoxin peroxidase (CpTPx) in C. parvum increased after a high dose of γ-irradiation to the parasite. CpTPx is a two-cysteine peroxiredoxin that contains cysteines at positions 49 and 170. Recombinant CpTPx fused to an N-terminal hexahistidine sequence, (His)(6)-CpTPx, exhibited substantial thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation in vitro. (His)(6)-CpTPx was used to screen sera from C. parvum-infected mice and humans for antibodies against CpTPx. In Western blots, 10% of the mouse sera and 20% of the human sera reacted with (His)(6)-CpTPx, suggesting that after infection by C. parvum CpTPx can induce a host-immune reaction but is not a major antigen. Immunolocalization studies revealed that CpTPx is expressed mainly in the cytoplasm of C. parvum at various developmental stages.

Comparison of resistance to γ-irradiation between Cryptosporidium parvum and Cryptosporidium muris using in vivo infection.

In the genus Cryptosporidium, there are more than 14 species with different sizes and habitats, as well as different hosts. Among these, C. parvum and C. hominis are known to be human pathogens. As C. parvum can survive exposure to harsh environmental conditions, including various disinfectants or high doses of radiation, it is considered to be an important environmental pathogen that may be a threat to human health. However, the resistance of other Cryptosporidium species to various environmental conditions is unknown. In this study, resistance against γ-irradiation was compared between C. parvum and C. muris using in vivo infection in mice. The capability of C. muris to infect mice could be eliminated with 1,000 Gy of γ-irradiation, while C. parvum remained infective in mice after up to 1,000 Gy of γ-irradiation, although the peak number of oocysts per gram of feces decreased to 16% that of non-irradiated oocysts. The difference in radioresistance between these 2 Cryptosporidium species should be investigated by further studies.

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium.

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.