Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite

Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 +/- 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO(2) at 37 degrees C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the large oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog.     

Detection of Feline calicivirus (FCV) from Vaccinated Cats and Phylogenetic Analysis of its Capsid Genes

We analysed genogroups of four feline calcivirus (FCV) isolates (FCV-S, H10, Ao198-1 and ML89) obtained from cats that experienced FCV infection after having been vaccinated against FCV. New PCR primer sets (8F/8R, Ao-S/Ao-A, cp-S/cp-A) were also designed, since the conventional Seal primer failed to amplify the target sequences in two samples. The genogroups of the four isolates as well as eight global and 17 domestic strains were determined by phylogenetic analysis of their amino acid sequences. One out of the four strains (25%) isolated in this study, H10, was grouped into genogroup I, along with the vaccine strains F9 and FCV-255. The other three isolates (75%) belonged to genogroup II. Thus, there were more isolates in genogroup II than in genogroup I. However, the antibody values of the four isolates against cat anti-F9 antisera were significantly decreased. There may be no relationship between the neutralizing antibody titre and genogroup. Amino acid sequence alignment of the four isolates showed that only a single amino acid in region C, which is involved in neutralization epitopes, was different in ML89 strain from that of F9. The other three strains, H10, Ao198-1 and FCV-B, shared the same amino acid sequence with F9. Alignment of amino acids for linear epitopes in the F9 strain, which are located at regions D and E, showed variations in 5' hypervariable region (HVR) of E, whereas D and conE had only synonymous substitutions i.e. no change in the amino acid sequence. This mutation in 5' HVR of region E suggested a vaccine breakdown, as the region is known to be essential for antigenicity. The genogroup II FCV is likely to be the cause of the FCV infection in this study, while the vaccine strains belong to genogroup I. Thus, the existing vaccine may need reevaluation for its effectiveness.     

Variations in nitrogen uptake and nitrate-nitrogen concentration among sorghum groups

Nitrogen uptake and nitrate-N concentration in forage sorghums, which are related to ground water pollution or feed quality under conditions of crop fertilization by only animal wastes, were examined. Seventy-four genotypes of sorghum and Sudan grass were tested. They were classified into 4 groups; grain type and dual purpose type sorghums (6 and 13 genotypes, respectively, Sorghum bicolor Moench), sorgo type sorghum (21, S. bicolor), Sudan type sorghum (22, S. bicolor × S. sudanense (Piper) Stapf), Sudan grass (12, S. sudanense). There was a strong correlation between dry matter (DM) yield and N uptake, with the sorgo type producing the highest DM matter yield and showing the highest N uptake. Nitrate-N, which causes nitrate poisoning of ruminants, was detected mostly in the stem of all the genotypes. The nitrate-N concentration based on DM could be calculated accurately by multiplying the nitrate-N concentration of stem on a fresh matter basis by the DM partitioning ratio of stem divided by the DM concentration of stem. The grain type and the dual purpose type of sorghums with dry stem showed the lowest nitrate-N concentration because these plants had a lower DM partitioning ratio of stem and higher DM concentration of stem. Sudan grass and Sudan type sorghum with the genetic background of Sudan grass showed higher nitrate-N concentrations owing to their ability to accumulate nitrate-N.