Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3×10⁶ U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.     

Long-term management of a glaucomatous eye in a dog treated with medical therapy alone.

An acute open angle glaucoma of the right eye was diagnosed in an 8-year-old male Yorkshire terrier which was presented with anorexia, depression, and trembling. Abnormal findings of the right eye on admission included elevated intraocular pressure (IOP; 40 mmHg), the presence of fibrin and flare in the anterior chamber, and immature cataract. Morphological abnormalities of the iridocorneal angle were not detected, and an open angle was seen in the eye. Although an elevated IOP was observed at one year after admission, lowering IOP (< or = 24 mmHg) was maintained with medical therapy using dichlorphenamide (DCPA) and timolol maleate, DCPA alone, or no-treatment for 1973 days. This case suggests that lower IOP can be maintained with medical therapy alone for a long period in a patient with open angle glaucoma.     

Studies on the live-virus vaccine against infectious laryngotracheitis of chickens. II. Evaluation of the tissue-culture-modified strain C7 in laboratory and field trials

Laboratory and field tests were conducted to evaluate the immunogenicity of a live-virus vaccine against infectious laryngotracheitis (ILT) of chickens that was prepared using tissue-culture-modified strain C7. Eighty-three percent or more of chickens vaccinated by the ocular (OC) or intranasal (IN) route with 10(4.75) TCID50 of the attenuated strain C7 at 50 days of age were protected against challenge with a virulent strain of ILT virus without showing any clinical signs for 4 weeks post-vaccination (PV). When vaccine was administered by aerosol, however, only 65% of vaccinated chickens were protected against challenge. Fifty-seven percent of chickens vaccinated at 70 days of age maintained immunity for 6 months PV. Immune response in younger chickens was inferior to that in older ones. In the field trials, clinical observation revealed no adverse reactions caused by the vaccination, and 60% or more of broilers and 80% or more of layers vaccinated by the OC route were protected against challenge at 4 weeks PV. These results confirmed the safety and efficacy of the vaccine.     

Pathogenicity of infectious laryngotracheitis virus as measured by chicken embryo inoculation

A comparison was made between pathogenicities for chicken embryos of unattenuated and attenuated strains or isolates of infectious laryngotracheitis (ILT) virus. All 11-day-old chicken embryos inoculated with 10(3.0) or 10(4.0) TCID50 of unattenuated strain NS175 via allantoic cavity died within 6 days. On the contrary, no chicken embryos of the same age died when inoculated with the same amount of cell-culture-attenuated isolate C7 in a like manner. The mortality index for chicken embryos (MICE) was obtained by dividing the cumulative number of embryos dying within 7 days by the cumulative number of embryos surviving 7 days. The reliability of the MICE test was confirmed by duplicate and triplicate experiments with strain NS175 and isolate C7. MICE obtained in the experiments with 9 different strains or isolates of ILT virus ranged from 0 to 1, and the values were well correlated with the pathogenicities for chickens. The results from the present work suggest that strains or isolates with MICE less than 0.16 would have low or no pathogenicity for chickens, and those with MICE more than 0.27 would be highly pathogenic. Further studies are needed using additional isolates of ILT virus with varied pathogenicities.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.