Effects of Calcium Lactate on the Development of Chicken Embryos in a Shell-less Culture System up to Day Seventeen of Incubation

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.     

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.     

GM1 gangliosidosis in shiba dogs

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid beta-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a beta-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of beta-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

Spatially distributed morphological characteristics of macropores in forest soils of Hitachi Ohta Experimental Watershed, Japan

Morphological characteristics of macropores in forest soil profiles were investigated at Hitachi Ohta Experimental Watershed in Japan. Nine individual profiles at different locations (various spatial scales in a catchment) and twenty profiles at one site (a small spatial scale) were excavated to the bedrock to investigate density, origin, diameter, direction, and gradient of macropores. Macropore densities in a soil profile ranged from 3.5 to 29.1 per m and from 5.4 to 75.1 per m², respectively. Subsurface erosion, root channels, and interactions between subsurface erosion and root channels accounted for 36.9, 36.5, and 19.0%, of the described macropores. The mean macropore diameter in organic-rich soil layer (17–20 mm) was larger than in the B horizon (11–14 mm) at both spatial scales. The dominant gradients of all macropores in the organic-rich soil layer and B horizon were at negative oblique angles. Approximately 90% of the macropores in the organic-rich soil layer and approximately 80% of the macropores in the B horizon fell within the range between −50 and 50 degree planar direction. Subsurface flow and root systems are believed to play important roles in determining the morphological characteristics of macropores. These characteristics appear to have variable influences in different soil horizons rather than at different spatial scales. A part of this paper was presented at the 103th (1992) and 105th (1994) Annual Meetings of Japanese Forestry Society.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.