Effect of polymorphism in egg white lysozyme on muramidase and antibacterial activities as well as hatchability in the Japanese quail (Coturnix japonica)

Lysozyme is one of the best characterized antimicrobial proteins in egg white. Three phenotypes of egg white lysozyme in Japanese quail, Coturnix japonica, (namely fast; slow; and the combination, FS) were observed by acid polyacrylamide gel electrophoresis. The fast phenotype showed faster mobility on Acid-PAGE than the slow phenotype. Comparison of the coding sequences for lysozyme derived from the slow and fast phenotypes revealed a nonsynonymous SNP at nucleotide position 115 from the translation initiation site, which alters AA sequence of lysozyme. This nonsynonymous SNP converted glutamine (Q) in the slow phenotype to lysine (K) in the fast phenotype at AA residue 21 of mature lysozyme (Q21K). Here, we investigated the effect of these phenotypes on muramidase activity, antibacterial activity, and hatchability. Muramidase activity toward isolated cell walls of Micrococcus lysodeikticus was in the order: fast allozyme > slow allozyme > chicken (Gallus gallus), but no significant difference was found among the 3 (P > 0.05). Antibacterial activity against live Staphylococcus aureus cells was significantly greater for the fast allozyme than the slow allozyme from 20 h after incubation (P < 0.05). For the antibacterial effects against live Escherichia coli cells, the activity of fast was significantly higher than that of slow at 16 h after incubation (P < 0.05). Hatchability was estimated for reciprocal crosses of Japanese quail with the FF (fast) and SS (slow) genotypes. Hatchability was 92.5% in FF male × SS female crosses and 87.2% in SS male × FF female crosses. A Cochran-Mantel-Haenszel test revealed a significant difference between the crosses (P < 0.05) and indicated that the female-derived slow phenotype led to improved rates of hatching. Our results suggest that the nonsynonymous SNP in Japanese quail lysozyme influences the electrophoretic migration, muramidase activity, and antibacterial activity of the protein, in addition to the hatchability of the eggs. These results demonstrate, for the first time, a significant difference in antibacterial activity and hatchability between 2 lysozyme phenotypes in Japanese quail.     

Lysis of myelocytes in chickens infected with infectious bursal disease virus

In specific-pathogen-free chickens infected with the highly virulent HPS-2 strain or virulent reference GBF-1 strain of infectious bursal disease virus (IBDV), pathologic changes of the bone marrow were investigated. On histologic examination, bone marrow lesions were prominent in the HPS-2 group but only mild in the GBF-1 group. The bone marrow of the HPS-2 group showed severe lysis and depletion of heterophil myelocytes with pyknotic nuclear alteration 2-3 days after inoculation. On examination with an electron microscope, heterophil myelocytes were characterized by shrinkage of the cytoplasm and peripheral condensation of nuclear chromatin. IBDV particles were not detected in altered myelocytes. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method demonstrated a positive reaction in only heterophil myelocytes. In contrast, nucleosomal DNA fragmentation in HPS-2-infected bone marrow cells was indiscernible by agarose gel electrophoresis. These findings indicate that lysis of bone marrow cells is selectively induced in heterophil myelocytes at an early stage after IBDV infection and independent of virus replication.     

Adenomyosis with severe inflammation in the uterine cervix in a dog

A dog with purulent vulval discharge was found to have many cysts containing purulent mucus in the uterine cervix. The uterine horns did not contain any pus. Histological examination revealed a cervical adenomyosis infiltrated by many neutrophils. After an ovariohysterectomy including the whole cervix, the dog recovered well.     

Temporal dynamic changes in synthesis of chondroitin sulfate isomers in canine articular chondrocyte culture

To investigate temporal dynamic changes in the synthesis of chondroitin 6-sulfate (CS6) and chondroitin 4-sulfate (CS4) in vitro, normal articular cartilage of femoral heads was harvested from three dogs. Chondrocytes were isolated and cultured in alginate microspheres for 21 days. On days 7, 14 and 21, DNA content was quantified by fluorometric assay using Hoechst 33258. On days 14 and 21, proteoglycans were extracted, and the amounts of CS6 and CS4 were quantified after chondroitinase ABC digestion using capillary electrophoresis. The DNA content and amounts of CS6 and CS4 increased during the culture period. The amounts of CS6 and CS4 divided by DNA content revealed that the synthesis of CS6 was more up-regulated than CS4.     

Relationship between liver disorders and protection against Eimeria stiedai infection in rabbits immunized with soluble antigens from the bile of infected rabbits

Soluble antigens exist in the bile of rabbits infected with Eimeria stiedai (E. stiedai) in the acute phase, and rabbits immunized with the antigens show resistance against the infection. In this study, the liver function of rabbits immunized either with the soluble antigens or PBS were examined following the parasite challenge. Rabbits immunized with PBS shed a number of oocysts and showed an increase in r-glutamyltransferase (GGT) activity and a decrease in blood Indocyanine green (ICG) clearance. However, rabbits immunized with the soluble antigens shed a lower number of oocysts and showed a transient increase of alanine-aminotransferase (ALT) activity on Day 8 post-challenge (p.c.). The blood Indocyanine green clearance of the rabbits showed no change throughout the experiment. By histopathological observation of the liver, a number of merozoites were found in the biliary ducts on Day 8 post-challenge in the non-immunized rabbits. In contrast, a number of lymphocytes and neutrophilic leukocytes assembled around the biliary ducts of the immunized rabbits, but few parasites were found there on Day 8 post-challenge. These results suggest that the soluble antigens stimulate local immune reactions, for example around the biliary ducts, resulting in elimination of the parasite's development.     

Equine pyoderma associated with malnutrition and unhygienic conditions due to neglect in a herd

Twelve horses kept at a riding club suffered from pyoderma. All the horses displayed crusting, scaling and alopecia. The lesions were distributed in the chest, back, rump and limbs. Some of the horse patients also showed epilation with an attached crust similar to a 'paintbrush lesion' of dermatophilosis, but normal skin flora or opportunistic pathogenic bacteria were only isolated from the lesions. Some patients clearly showed weight loss, anemia and low levels of serum protein and cholesterol. General condition and skin lesions of the patients were improved gradually with improvement of feed and environment after being moved to new stables. Malnutrition under conditions of poor hygiene and poor management due to neglect might be associated with these equine cases of pyoderma in the herd.     

Characterization of some Asian isolates of Phytophthora infestans

A total of 401 isolates of Phytophthora infestans were collected from eight Asian regions (Korea, India, Taiwan, Indonesia, Thailand, Nepal, China and Japan) between 1992 and 2000 – 318 from potato and 83 from tomato. The isolates were analysed for mating type, metalaxyl resistance, RG57 fingerprinting, mitochondrial DNA (mtDNA) haplotype and the polymorphism of three allozyme loci, i.e. glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ) and malic enzyme ( Me ). The isolates were multilocus-genotyped based on RFLP (RG57) fingerprint, dilocus allozyme genotype, mtDNA haplotype and mating type. Twenty multilocus genotypes were identified among 125 isolates. Of these genotypes, 14 had not been previously reported. Some of the multilocus genotypes were common to isolates from several geographical regions, suggesting migration. The metalaxyl-resistant isolates belonged to the multilocus genotypes JP-1, JP-2, and JP-3. Multilocus genotypes coexisting in a single field were found in following regions: Thailand (1994), central China (1996), Nepal (1997) and Japan (1998 and 2000). The possible origins of certain genotypes are discussed, including the possibility of sexual recombination within the P. infestans populations in Nepal and perhaps Thailand.     

Lesional expression of thymus and activation-regulated chemokine in canine atopic dermatitis

Glucocorticoids are reported to bias cytokines to a Th2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs. The current study was conducted to revisit the issue of glucocorticoid modulation of Th1/Th2 cytokine production and evaluate migration inhibitory factor (MIF) mRNA expression in cultured pig splenocytes treated with physiologically relevant concentrations of cortisol (CORT). Dexamethasone (DEX) was included for comparison. In Experiment 1, DEX, at 150 and 300 nM, suppressed concanavalin (ConA)-stimulated IFNgamma at both 12 and 24 h in culture, and IL-10 at 24h (P<0.05). Both 150 and 300 nM CORT suppressed IL-10 at 24 h (P<0.05), but neither concentration affected IFNgamma at 24 h. In Experiment 2, cells were cultured with a broader range of CORT for 48 h following ConA. Parallel cultures with identical treatments also were conducted in separate plates for evaluation of glucocorticoid regulation of MIF mRNA. IFNgamma was reduced by 300 nM DEX at 12, 24, and 48 h (P<0.05), whereas 150 and 300 nM CORT blunted IFNgamma at 24 h (P<0.05), but not 48 h. ConA increased IL-2 (P<0.01), but none of the steroid treatments affected IL-2. At both 12 and 24 h, IL-10 was reduced by 300 nM DEX and by 150 and 300 nM CORT (P<0.05). ConA increased relative abundance of MIF mRNA (P<0.001), but no steroid treatment affected MIF mRNA. In Experiment 3, steroid additions were delayed by 24 h after ConA, and cytokine concentrations evaluated 48 h later. Again, separate cultures were used for determination of effect of treatments on MIF mRNA. None of the steroid treatments affected IFNgamma, but 300 nM DEX reduced IL-10 (P<0.05). All of the CORT treatments (75-300 nM) reduced MIF mRNA (P<0.05), whereas DEX did not affect MIF mRNA in this experiment. The current experiments suggest that both DEX and high physiological concentrations of CORT can suppress both type 1 and type 2-like cytokines in cultured pig splenocytes. But, IL-10 was generally more sensitive to CORT suppression with increased time in culture than was IFNgamma. In addition, MIF mRNA could be suppressed by delayed addition of CORT to porcine splenocytes. Taken together, the data do not support the hypothesis that CORT directs the cytokine milieu toward a type 2 bias in cultured pig splenocytes.     

Immunohistochemical and ultrastructural identification of Fusobacterium necrophorum subsp. necrophorum in bovine fatal necrotizing glossitis

A 37-day-old male Japanese black calf showing marked salivation and leucocytosis died and was examined the tissues histologically. Histological lesions were characterized by severe focal necrotic glossitis on the ventral side of the root of the tongue. Immunohistochemically, Fusobacterium necrophorum subsp. necrophorum antigen was detected in the necrotic tissues and its distribution corresponded to that of the gram-negative, nonsporeforming, long filamentous organisms. Ultrastructural similarities between the organism and F. necrophorum subsp. necrophorum, but not subsp. funduliforme were observed. These findings clearly demonstrated that the fatal necrotic glossitis was caused by F. necrophorum subsp. necrophorum. This is the first report of bovine fatal necrotizing glossitis with leucocytosis caused by F. necrophorum subsp. necrophorum infection, and this organism may be an important fatal pathogen in calves with glossal lesions.     

Distribution of blastodermal cells transferred to chick embryos for chimera production using windowed eggs

1. To improve the production of chimeras, the distribution of donor blastodermal cells after transferring into recipient embryos was examined morphologically.

2. Donor blastodermal cells were distributed near the site of injection in the epiblast and in the subgerminal cavity and yolk. Some filled the hole made by the micropipette and were distributed outside the epiblast. Many were buried in yolk. In some cases, more donor blastodermal cells were located in the yolk than in the subgerminal cavity and some were located 800 μm below the under‐surface of the epiblast.

3. It is recommended that injection should be as shallow as possible to increase the proportion of chimeras produced, and that some means is needed to prevent blastodermal cells from escaping from the hole produced by injection.