Kameo Shimura

Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction

A method was developed for the discrimination of 8 Eimeria species of chickens, i.e., E. acervulina, E. brunetti, E. mitis, E. maxima, E. necatrix, E. praecox, E. tenella, and E. hagani using the 2-step polymerase chain reaction (PCR). In the first PCR, the small subunit ribosomal RNA (srRNA) gene was amplified from the parasite genome using conserved sequences for the Apicomplexa srRNA gene as the primers. The srRNA gene amplified from the parasite genome was discriminated in the second step by random-amplified polymorphic DNA (RAPD) PCR using 10 arbitrary primers. Each arbitrary primer produced species-specific RAPD patterns that provided a simple method for species identification from the srRNA genes of the 8 Eimeria species. This method should be useful for discrimination of the parasite species for diagnosis or epidemiological surveys of chicken coccidiosis.

High-throughput RNA sequencing profiles and transcriptional evidence of aerobic respiratory enzymes in sporulating oocysts and sporozoites of Eimeria tenella.

Seven species of Eimeria are responsible for coccidiosis in chickens. Eimeria tenella is one of the most pathogenic parasites since it is associated with high mortality and great economic impact. The life cycle of the parasite includes development in the environment and in the intestinal tract. We conducted RNA sequencing using a next generation sequencer to obtain transcriptome information from the sporulating oocysts, and sporozoites. We collected 2.8 million 75 bp reads of a short-tag sequence, and 25,880 contigs were generated by the Oases assembler. A Blastx search of GenBank databases revealed that 7780 contigs (30.1%) had significant homology with deposited sequence data (E-value <1e-6); among these contigs, 6051 contigs were similar to those of Toxoplasma gondii while only 513 contigs (6.6%) were similar to those of E. tenella. After an orthological analysis conducted with the UniProt database of T. gondii, 6661 contigs were distributed within the categories of cellular components (1528 gene categories), biological processes (861 gene categories), and molecular functions (241 gene categories). The significantly matched contigs contained high numbers of enzymes associated with glycolysis, TCA, and the pentose-phosphate pathway. Most of the enzymes, measured by quantitative reverse transcription-PCR, were up-regulated in sporulating stage. These results suggest that the intracellular carbohydrate amylopectin could be used as an energy source for ATP production including glycolysis and the pentose-phosphate pathway, which generates NADPH and pentoses. Our data also suggest that Eimeria might possess a partial or similar pathway to the TCA cycle essential for aerobic respiration. Furthermore, the newly annotated and non-annotated contigs might contain E. tenella-specific or novel sequences.

Reduced Lipopolysaccharide (LPS)-Induced Nitric Oxide Production in Peritoneal Macrophages and Inhibited LPS-Induced Lethal Shock in Mice by a Sugar Cane (Saccharum officinarum L.) Extract

A sugar cane extract (SCE) has been found to have an immunostimulating effect in several animals. Lipopolysaccharide (LPS) is known to induce endotoxin shock via the production of inflammatory modulators such as tumor necrosis factor (TNF)-α and nitric oxide (NO). We examined in the present study the effects of SCE on the TNF-α and NO production in LPS-stimulated mice peritoneal cells and the endotoxin shock in mice. The supplementation of SCE to peritoneal macrophages cultured with LPS resulted in a significant decrease in NO production. All the mice injected intraperitoneally with LPS and D-galactosamine (LPS+GalN) died within 24 h. However, a peritoneal injection, but no intravenous or oral administration, of SCE (500–1,000 mg/kg) at 3 to 48 h before the LPS+GalN-challenge resulted in a significantly improved survival rate. These results suggest that SCE had a protective effect on LPS-induced endotoxin shock via one of possible mechanisms involving the suppression of NO production in the mouse peritoneal cavity.

Goats as natural intermediate hosts of Hammondia hammondi

The infectivity of Hammondia hammondi (the G-8 strain) to goats was studied to determine the origin of this strain, because it was originally isolated from feces of a cat fed goat muscles. No clinical signs except fever were observed in kids after oocyst inoculation. When cat fed muscles of these kids, the cats excreted oocysts. The prepatent period was 7 to 9 days and the patent period was 6 to 12 days. From these experiments it became clear that the G-8 strain was infectious to goats. The origin of this strain is very probably the goat, which thus is one of the natural intermediate hosts of H. hammondi.

Synchronous development of Eimeria tenella in chicken caeca and utility of laser microdissection for purification of single stage schizont RNA.

Eimeria tenella is recognized worldwide as a significant pathogen in the poultry industry. However, a lack of methods for isolating developing schizonts has hindered the use of transcriptome analyses to discover novel and developmentally regulated genes. In the present study, we characterized the long-term successive development of E. tenella in infected chicken caeca and assessed the utility of laser microdissection (LMD) for the isolation of schizont RNA. Developmental stages, including those of the first, second, and third-generation schizonts and gametocytes, were synchronous. Using LMD, only the mature second-generation schizonts were successfully excised from the lamina propria, and non-degraded RNA was purified from the schizonts. E. tenella-specific genes were amplified by reverse transcription polymerase chain reaction (RT-PCR). These results augment our understanding of the E. tenella life cycle, and reveal LMD as a potentially useful tool for gene expression analyses of the intracellular stages of E. tenella.

Resistance of chicks against reinfection with Leucocytozoon caulleryi

Chickens were inoculated with five to 40 sporozoites of Leucocytozoon caulleryi at 1 to 9 days of age. Mortality, parasitemia, appearance of serum-soluble antigen, and antibodies were examined. Chickens that survived primary infection were reinoculated with 3 x 10(3) to 1 x 10(4) sporozoites and examined for resistance against reinfection by the same criteria used in primary infection. After primary infection, up to 80% of chickens died of leucocytozoonosis. Surviving chickens showed parasitemia from 13 days to 23 or 24 days after inoculation. Serum-soluble antigen and antibodies were detected in most chickens, except for some chickens that were infected at 1 to 3 days of age. After secondary infection, only a few chickens that received primary infection at 1 to 7 days of age showed a small number of parasites in their peripheral blood. Most of the other chickens were resistant to reinfection. Resistance to reinfection was recognized in some chickens that had not showed antibody production before secondary infection.

Localization of Eimeripain, an Eimeria tenella Cathepsin B-Like Cysteine Protease, during Asexual and Sexual Intracellular Development in Chicken Ceca

ABSTRACT Hemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.