High prevalence of Cryptosporidium infection caused by C. scrofarum and C. suis among pigs in Thailand.

Cryptosporidium spp. is an important intestinal protozoan causing diarrhea among both healthy and immunocompromised patients especially those with HIV/AIDS. Cryptosporidium spp. can be transmitted via foodborne, waterborne and person-to-person routes. In addition, several Cryptosporidium species are zoonotic. This study aimed to determine the prevalence of Cryptosporidium infection among pigs raised in both smallholder (<50 heads/farm) and large scale farms (50-500 heads/farm) in Chonburi Province, eastern Thailand using nested PCR amplifying the small subunit of the ribosomal RNA (SSU-rRNA) gene. DNA sequencing was also performed to identify the species of Cryptosporidium. A total of 245 fecal samples were collected from 11 pig farms. The overall prevalence of Cryptosporidium infection was 20.8% (51/245) which were found in both smallholder and small large scale pig farms. The prevalence of Cryptosporidium infection among pigs aged ≤6 months was significantly higher than those aged >6 months (p < .001). Among 51 Cryptosporidium positive samples, Cryptosporidium scrofarum (42/51, 82.4%) and Cryptosporidium suis (9/51, 17.6%) were identified. The prevalence of C. scrofarum infection observed among pigs aged ≤6 months was significantly higher when compared with those aged >6 months (20.7% and 2.1%, respectively, p < .001). The high prevalence of C. scrofarum and C. suis infections among pigs could be a potential source of infection to humans.

Prevalence and Molecular Characterization of Enterocytozoon bieneusi among Pigs in Chonburi Province, Eastern Thailand.

Enterocytozoon bieneusi is an organism that infects a wide variety of vertebrates, including humans. Pigs also harbor E. bieneusi, of which several genotypes have been recently detected in human feces. The aim of this study was to determine the prevalence of E. bieneusi infection among pigs raised in three smallholder farms and eight small large-scale farms in Chonburi Province, Eastern Thailand, using nested polymerase chain reaction of the internal transcribed spacer (ITS) of the small subunit of ribosomal RNA gene and to investigate genotypes of E. bieneusi isolates using nucleotide sequencing and phylogenetic tree analysis of the ITS region. Of 244 stool samples, E. bieneusi was detected in 14.8% (36/244). Two known zoonotic genotypes, that is, genotypes E (77.8%) and F (22.2%), were identified. Using phylogenetic tree analysis, these two genotypes were clustered in human pathogenic and zoonotic potential groups, designated as group 1. The high prevalence of zoonotic genotypes of E. bieneusi among pigs suggests that pig farming is one of the potential sources of human infection. This is the first report of E. bieneusi genotypes among pigs raised in pig farms in Eastern Thailand.

Identification of Blastocystis subtype 1 variants in the Home for Girls, Bangkok, Thailand.

A cross-sectional study of Blastocystis infection was conducted to evaluate the prevalence, risk factors, and subtypes of Blastocystis at the Home for Girls, Bangkok, Thailand in November 2008. Of 370 stool samples, 118 (31.9%) were infected with Blastocystis. Genotypic characterization of Blastocystis was performed by polymerase chain reaction and sequence analysis of the partial small subunit ribosomal RNA (SSU rRNA) gene. Subtype 1 was the most predominant (94.8%), followed by subtype 6 (3.5%) and subtype 2 (1.7%). Sequence analyses revealed nucleotide polymorphisms for Blastocystis subtype 1, which were described as subtype 1/variant 1, subtype 1/variant 2. Blastocystis subtype 1/variant 1 was the most predominant infection occurring in almost every house. The results showed that subtype analysis of Blastocystis was useful for molecular epidemiological study.

Regulation of CRIg expression and phagocytosis in human macrophages by arachidonate, dexamethasone, and cytokines.

Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg(+) macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-γ, IL-4, and transforming growth factor-ß1 decreased CRIg expression on macrophages. This down- and up-regulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation.

Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1.

BACKGROUND: Human antibodies that interfere with the biological activity of haemagglutinins (HAs) of influenza viruses have high potential as an antiviral agent. METHODS: Human single-chain antibody fragments (HuScFv) to recombinant and native HAs of the influenza virus H5N1 subtype were produced using a human antibody phage display library with the intention to increase the therapeutic arsenal against this highly pathogenic virus. RESULTS: The HuScFv inhibited HA activity and neutralized infectivity of both homologous and heterologous strains and clades of the H5N1 subtype in Madin-Darby canine kidney cell cultures. Intraperitoneally injected HuScFv also mediated immunotherapeutic protection in mice that were intranasally challenged with highly pathogenic H5N1 viruses belonging to different strains and clades. CONCLUSIONS: Our data indicate that it might be worth pursuing these HuScFv further for future consideration as candidates for influenza intervention and treatment.

Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand.

In January 2005, a survey of intestinal parasitic infections was performed in a primary school, central Thailand. Of 675 stool samples, Blastocystis was identified with a prevalence of 18.9%. Genetic characterization of Blastocystis showed subtype 1 (77.9%) and subtype 2 (22.1%). Study of the water supply in this school was performed to find the possible sources of Blastocystis. Blastocystis from one water sample was identified as subtype 1, which had a nucleotide sequence of small subunit (SSU) ribosomal RNA (rRNA) gene that was 100% identical to that of Blastocystis infected in schoolchildren. Our information supports the evidence of water-borne transmission in this population.

Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus.

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.

The usefulness of short-term in vitro cultivation for the detection and molecular study of Blastocystis hominis in stool specimens.

When in vitro cultivation was used as the 'gold standard' for the detection of Blastocystis hominis in stool specimens, simple smear and trichrome staining showed sensitivities of 16.7% and 40.2% and specificities of 94% and 80.4%, respectively. In vitro cultivation also enhanced PCR amplification for the detection of B. hominis in stool specimens. Our data show the usefulness of in vitro cultivation for the detection and molecular study of B. hominis in stool specimens.

Evidence of waterborne transmission of Blastocystis hominis.

A cross-sectional study was performed in February 2001 to evaluate the prevalence and risk factors of Blastocystis hominis infection in army personnel who resided in an army base in Chonburi, Thailand. A total of 904 army personnel were enrolled in this study. Short-term in vitro cultivation was used to detect B. hominis in stool samples. In this population, B. hominis was the parasite most frequently found, and was identified in 334 of 904 stool specimens (36.9%). A significant association between B. hominis infection and symptoms was identified that might emphasize the role of B. hominis as a human pathogen. After adjustment for potential confounders, significantly increased risk of being infection with B. hominis was associated with being a private, working in a specific unit, and consuming unboiled drinking water. Thus, waterborne transmission of B. hominis infection was indicated at this army base. However, other modes of transmission cannot be ruled out.

Blastocystis isolates from a pig and a horse are closely related to Blastocystis hominis.

Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. However, the contribution of zoonotic transmission remains unclear due to the absence of molecular proof of these organisms being identical to those found in humans. We report herein the similar subgroup of Blastocystis isolates from humans, pigs, and a horse using a restriction fragment length polymorphism (RFLP) analysis of partial small-subunit ribosomal DNA (ssu rDNA). Additionally, sequence and phylogenic analysis of partial ssu rDNA of Blastocystis from a human, a pig, and a horse sharing a common subgroup shows that Blastocystis isolates from a pig and a horse were monophyletic and closely related to B. hominis, with 92 to 94% identity. These results suggest the possibility of zoonotic potential of Blastocystis.