1NM-PP1 treatment of mice infected with Toxoplasma gondii

Bumped kinase inhibitors (BKIs) target analog-sensitive kinases, which the genomes of mammals rarely encode. Previously, we demonstrated that a BKI effectively suppressed the in vitro replication of Toxoplasma gondii, the causative pathogen of toxoplasmosis, by targeting T. gondii calcium-dependent protein kinase 1 (TgCDPK1) (Eukaryotic Cell, 9: 667-670). Here, we examined whether the BKI 1NM-PP1 reduced parasite replication in vivo. A high dose of 1NM-PP1, by intraperitoneal injection, just before the parasite inoculation effectively reduced the parasite load in the brains, livers, and lungs of T. gondii-infected mice, however, a low dose of 1NM-PP1 with oral administration didn't change the survival rates of infected mice.     

Seasonal change in the number of Cryptosporidium parvum oocysts in water samples from the rivers in Hokkaido,Japan, detected by the ferric sulfate flocculation method

An epidemiological study was carried out in natural water supplies of Hokkaido, one of the largest dairy prefectures in Japan. To investigate the prevalence of Cryptosporidium parvum (C. parvum) oocysts water samples were collected from three rivers in the eastern area of Hokkaido from August 1999 to October 2001, and C. parvum oocysts were collected and purified by the ferric sulfate flocculation method. The oocysts were detected using the immunofluorescent assay test (IFAT) and 4', 6-diamidino-2-phenylindole (DAPI) staining. The seasonal change in the number of oocysts detected was observed. Oocysts increased in numbers from the late summer to the early autumn (from August to November), thereafter, they exhibited a trend to decrease until December, when no oocysts could be detected. The maximum number of oocysts detected in the three rivers was 3.50, 5.00 and 3.33 oocysts/l, respectively. The oocyst density in river water changed in relation to the season in 1999, 2000 and 2001. This report first cleared up the seasonal changes in C. parvum oocysts number in river water.     

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido,Japan

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.     

Characterization of Env antigenicity of feline foamy virus (FeFV) using FeFV-infected cat sera and a monoclonal antibody

To characterize neutralizing antigenicity in relation to env genotypes of feline foamy virus (FeFV), serological analyses were performed using FeFV-infected cat sera and several field isolates including two env genotypes (F17- and FUV-types). Since three cats from which FeFV were isolated were found to have undetectable titers of virus neutralization (VN) antibodies, even to the homologous virus, VN antibodies were further examined with complement supplementation as an enhancement factor. With the presence of complement, the VN titers of FeFV-infected cat sera increased drastically. Although most of serum samples neutralized strains of either env genotype, sera sampled from two cats neutralized all the strains examined at similar titers, suggesting that superinfection with both env genotypes of FeFV might have occurred in the two cats. Further, we produced a monoclonal antibody (mAb) specifically neutralizing FeFV strains of FUV-type. The mAb was shown to have higher affinity to an epitope on Env of FUV-type than that of F17-type by immunoprecipitation assay. This study supplies basic information important for studies on FeFV vector development as well as on the relationship between the virus and the host immune response.     

Comparison of the polymerase chain reaction-restriction fragment length polymorphism pattern of the fiber gene and pathogenicity of serotype-1 fowl adenovirus isolates from gizzard erosions and from feces of clinically healthy chickens in Japan.

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.