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.     

Cross-reactivity between human and feline Ia antigens, using a monoclonal antibody HLA-D.m1

A murine monoclonal antibody HLA-D.m1 produced against human Ia antigens which react with 10% to 30% of human peripheral-blood mononuclear cells was found to react with greater than 90% of feline mononuclear cells. The antigen was not expressed on granulocytes. By direct immunofluorescence, 7% to 41% of feline lymphocytes expressed surface immunoglobulins. In mixed-lymphocyte reaction, the addition of antibody HLA-D.m1 to cultures abolished proliferation. These studies indicate that most feline peripheral-blood mononuclear cells express Ia-like antigens.     

Teratoma of the umbilical cord in a giraffe (Giraffa camelopardalis reticulata)

A 12-year-old pregnant female giraffe (Giraffa camelopardalis reticulata) died approximately 2 months prior to her anticipated parturition date. At necropsy, a mass measuring approximately 20 x 36 x 20 cm was observed, attached to the umbilical cord, the latter being otherwise normal in appearance. Histologically, the mass contained 3 germinal tissue components with areas of squamous epithelium, respiratory epithelium, primitive neural tissues, glial tissue, peripheral nerve, adipose tissue, cartilage, and smooth muscle. Based on these findings, the tumor was diagnosed as a teratoma originating from the umbilical cord. This is possibly the second reported case of umbilical cord teratoma in animals.     

Localization of interleukin-6 receptor mRNA in the pregnant and non-pregnant mouse uterus

To understand roles of interleukin 6 (IL-6) family cytokines for pregnancy in mice, localization of IL-6 receptor (IL-6R) mRNA was investigated in non- and early pregnant uteri by in situ hybridization. IL-6R mRNA was expressed in all non-pregnant uteri and in pregnant uteri from the third day (Day 3) to the sixth day of pregnancy (Day 6; the day of plug = Day 1). IL-6R mRNA signals were detected in non-pregnant mice in the luminal and glandular epithelium. Signal strength varied according to the sexual cycle. There was no correlation between the signal strength of the IL-6R mRNA and the serum concentrations of progesterone and 17beta-estradiol, which show a monophasic rise in the non-pregnant sexual cycle. In pregnant mice, slight signals were detectable in the luminal and glandular epithelium on Day 3. IL-6R mRNA messages increased with progression towards Day 4, however, localization changed drastically on Day 5. Stromal cells abruptly expressed their mRNA on Day 5, and these cells strongly expressed it on Day 6. The function of IL-6R in the luminal and glandular epithelium might be different from that in the stroma during the implantation period. In addition, few signals were identified in the stromal cells adjacent to the luminal epithelium on Day 6. This suggests that there are two types of stromal cells on Day 6 in mice.     

Enzyme-linked immunosorbent assay for the detection of canine Leptospira antibodies using recombinant OmpL1 protein

OmpL1 is a 31-kDa outer membrane protein characterized in 1993 and known to be expressed only in pathogenic Leptospira spp. Recombinant OmpL1 (GST-rOmpL1) was expressed for use as an ELISA antigen for the detection of anti-Leptospira antibodies. In immunoblot analysis, the protein reacted with sera of dogs infected with three different serotypes of Leptospira interrogans, while did not react with sera of dogs both uninfected negative controls and infected with Borrelia burgdorferi, which is closely related to Leptospira spp. Moreover, in ELISA using GST-rOmpL1, the optical density (O.D.) values from the positive controls were very high (1.125 +/- 0.549). In contrast, the O.D. values from clinically healthy dogs and dogs with diseases other than leptospirosis were very low (0.109 +/- 0.046 and 0.089 +/- 0.046, respectively). These data suggest that the detection of anti-Leptospira antibodies by ELISA using the GST-rOmpL1 protein can be applied for diagnosis of canine leptospirosis.     

Retrospective serological analysis of spontaneous CDV infection in 192 dogs

Spontaneous cases of canine distemper virus (CDV) infection were serologically evaluated. The 192 dogs in which CDV antigen was confirmed from tonsil by immunohistological examination were 2- to 4-months old, of various breeds, and unvaccinated. The prognoses were good in 74 dogs with significantly high levels of anti-CDV passive hemolytic aggregation (PHA) titer. In the other 118 dogs with poor prognoses, anti-CDV PHA titer was not detected. Anti-CDV PHA titer had the most significant association with the prognoses of CDV infection, and could be the most reliable and useful indicator for evaluating such prognoses.     

Spontaneous Glomerular and Tubulointerstitial Lesions in Common Marmosets (Callithrix jacchus)

Spontaneous progressive nephropathy dominated by glomerular lesions in common marmosets has been reported. However, the histopathologic characteristics, including the relationship between glomerular and tubulointerstitial lesions, have not been described in detail. In the present study, the authors examined the histopathologic characteristics of the background renal lesions in common marmosets (3 males and 9 females, 3 to 8 years old). The severity of glomerular lesions was graded into 3 classes: grade I, no alteration; grade II, hilar/focal increase of mesangial matrix; grade III, global/diffuse increase of mesangial matrix. Tubulointerstitial lesions (tubular regeneration and hyperplasia and interstitial inflammation and fibrosis) were scored according to the area of each lesion. The renal lesions were characterized by enlargement of glomeruli, expanded mesangial area with increase of periodic acid-Schiff reaction-positive matrix, tubular regeneration and hyperplasia, and interstitial inflammation and fibrosis. Glomerular lesions progressed with increasing mesangial matrix and aging. Additionally, the tubulointerstitial lesions became exacerbated with progressing glomerular lesions. Tubular hyperplasia was divided into 4 types according to the structure of the cell layer (simple or stratified-like), the area of increased lining cells (partial or entire), cytoplasmic staining (eosinophilic or basophilic), brush border and thickness of basement membrane, and the activity of cell proliferation. In conclusion, the background renal lesions in common marmosets were characterized by glomerular lesions with increase of mesangial matrix, which progressed with aging, and secondary tubulointerstitial lesions, including tubular hyperplasia. Those lesions were thus diagnosed as progressive glomerulonephropathy in common marmosets.     

Indices using external measurements for assessing fat deposition of adult feral raccoons (Procyon lotor) in Kanagawa Prefecture, Japan

We examined the use of external measurements and relative fat deposition of adult feral raccoons (Procyon lotor) to develop relative indices of body fat deposition in post-growth feral raccoons. From March 2006 to March 2010, 288 adult raccoon carcasses (110 males, 178 females) collected in Kanagawa Prefecture, Japan, which were determined to be 24 months old, were subjected to external measurements of body weight (BW), girth measurement (GM), and body mass index (BMI). To assess relative body fat deposition, we visually classified abdominal subcutaneous fat into three grades (Visible Fat Index [VFI]: I-III). Significant differences in the means of BW (both sexes:P<0.01), GM (females: P<0.05, males: P<0.01), and BMI (both sexes: P<0.01) were detected between seasons. Notably, the means of BW, GM, and BMI (all, both sexes: P<0.01) differed significantly between VFI grades. However, by discriminant analysis with BW, GM, and BMI as independent variables, we obtained a significant discriminant function (both sexes: P<0.01) for distinguishing VFI I from higher VFI grades, but no significant equation was obtained for distinguishing between VFI II and VFI III. Based on the obtained structure matrix of discriminant analysis, BMI was the most valuable component for the discrimination of VFI grades. Thus, we conclude that BMI is a suitable complementary index for assessing relative body fat deposition of adult feral raccoons in Kanagawa Prefecture and may be generalizable to populations in other areas.