During pregnancy, a population of uterine NK cells, commonly called granulated metrial gland (GMG) cells, differentiates in the uterus of both immune competent and various immunodeficient mice. Regulatory mechanisms controlling the differentiation of GMG cells are not fully known. It has been proven that GMG cells are derived from bone marrow, appear under the influences of progesterone and estrogen, do not require the presence of an embryo, and are associated in rodents with decidualization of the uterine stroma. Mice of genotype aly/aly are genetically deficient in lymph nodes and Peyer's patches due to a lymphoid-associated mesenchymal disorder. They are considered to be a useful model for the study of interactions between lymphocytes and stromal components. This immunodeficient animal is completely different from nu/nu and scid/scid mice who differentiate GMG cells during pregnancy. To determine whether the differentiation of GMG cells depends on mesenchymal interactions in the uterus, aly/aly mice were studied histologically between days 10 and 14 of pregnancy for differentiation of GMG cells and development of the metrial gland. Metrial gland tissue was present and appeared normal in aly/aly mice. There were no significant differences in the distribution of GMG cells in comparison to control pregnant aly/+ mice. Fewer GMG cells were present in aly/aly mice than aly/+ mice on days 12 and 14 of pregnancy. The features of individual GMG cells were different on days 10 and 12 of pregnancy. GMG cells in aly/aly mice were small in size and the granules were poorly developed. By day 14, however, GMG cells acquired a mature size and the granules appeared mature. It is likely that GMG cell differentiation was delayed in pregnant aly/aly mice, due to a mesenchymal disorder affecting metrial gland development in this animal.
The growth pattern of somatostatin cells was clarified immunohistochemically from day 12 to day 18 of gestation in the rat fetus. On day 12, somatostatin cells first appeared within the pancreatic anlage. The total number of somatostatin cells was gradually increased from day 12 to day 16 and rapidly increased thereafter. It may be concluded that such a sequence of events of development in somatostatin cells occurs in a fashion similar to that in B cells.
Although isolation of Rhodococcus equi from tracheobronchial aspirates is thought to be a definitive diagnosis of R. equi pneumonia in foals, virulence of isolates from the aspirates of infected foals remains obscure. In the present study, transtracheal aspirates were collected from thirty-one 1-to 6-month-old foals, which showed clinical signs of respiratory tract infection, and R. equi isolates were analyzed for the presence of virulence plasmids and virulence-associated antigens. Moreover, this method was compared with a serodiagnosis by an enzyme-linked immunosorbent assay (ELISA) to evaluate the sensitivity of the ELISA. Of the 31 foals, 21 revealed positive cultures for R. equi. Of the 21 foals, 20 (95%) had an ELISA OD value of 0.3 (positive limit of this test) or higher at the initial medical examination. All of the isolates from the aspirates were virulent R. equi, which contained virulence plasmids and expressed virulence-associated antigens. In the remaining 10 foals showing a negative culture for R. equi, 3 foals had positive ELISA titers. Six foals died during the treatment, and necropsy revealed that 5 of the 6 foals had R. equi infection characterized by large abscesses in the lungs, and 3 of the 5 foals also had intestinal lesions. All clinical isolates from the lesions of the foals were virulent R. equi. These results support the assumption that isolates from the transtracheal aspirates of infected foals are virulent R. equi and the sensitivity of ELISA might demonstrate a serodiagnostic value for early diagnosis of R. equi infection in foals.
The emigration of leukocytes from calves with β2 integrin deficiency (BLAD) into bronchoalveolar spaces and scraped tissues was compared to that of normal calves. Polymorphonuclear neutrophils were found in bronchoalveolar lavage fluid from BLAD-affected calves showing chronic pneumonia. The neutrophils were complement receptor type 3 (CR3)-negative when characterized by flow cytometric analysis using anti-CD18 monoclonal antibody. Chemiluminescent response mediated by CR3 in neutrophils isolated from bronchoalveolar lavage fluid from BLAD-calves showed similar findings obtained from CR3-deficient neutrophils. Neutrophils from normal calves migrated into scraped tissue which was prepared in an upper gluteal surface area, whereas few leukocytes from calves with BLAD migrated to the scraped tissue, evaluated by skin window (Rebuck) method. These findings confirmed the extravasation of CR3-deficient leukocytes into bronchoalveolar lumen in BLAD calves, and demonstrated in vivo characteristics of extravasating property of normal and CR3-deficient neutrophils into scraped tissues.
In order to evaluate the usefulness of liposomes as oral vaccines, the stability of liposomes and serum IgA antibody response to antigen associated with liposomes after oral administration were examined. Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), and cholesterol (Chol) (1:1:2, molar ratio), distearoylphosphatidylcholine (DSPC) and Chol (7:2, molar ratio), and DSPC, DPPS, and Chol (7:3:2 or 1:1:2, molar ratio) were stable in acidic solution (pH 2.0), bile, and pancreatin solution, whereas liposomes composed of DPPC and Chol (7:2, molar ratio) and DPPC, DPPS, and Chol (7:3:2, molar ratio) were unstable in pH 2.0 and/or bile solutions. After the oral immunization of antigen (ganglioside GM1)-containing liposomes composed of DPPC, DPPS, and Chol (1:1:2, molar ratio) to mice, the serum IgA antibody responses against ganglioside GM1 were found. Furthermore, when monophosphoryl lipid A was incorporated into liposomes containing ganglioside GM1, further augmentation of IgA responses to ganglioside GM1 was observed. On the other hand, the oral administration with liposomes composed of DPPC, Chol, and ganglioside GM1 (unstable liposomes), ganglioside GM1 mixed with liposomes composed of DPPC, DPPS and Chol, and ganglioside GM1 alone was unable to induce any detectable anti-ganglioside GM1 IgA antibody responses. These results suggest that liposomes which showed the stability to acidic solution, bile, and pancreatin solution would serve effectively as an oral delivery vehicle for inducing mucosal immune responses.
The effect of ecabapide, a novel gastroprokinetic agent, on gastric emptying was examined in stroke-prone spontaneously hypertensive rats (SHRSP), which are known to exhibit gastric malfunctions as a result of autonomic nervous system disorder. Ecabapide was administered orally to SHRSP in various dose-regimens. Gastric emptying was evaluated by the acetaminophen (APAP) method using semi-solid meal under the conscious conditions. Ecabapide significantly enhanced gastric emptying of SHRSP at 1 mg/kg in single administration, 0.3 and 1 mg/kg in 2-week treatment and 7.7 mg/kg in 4-week (diet) exposure. These results suggest that the effect of ecabapide is exerted without showing tachyphylaxis.
Nucleotide sequences surrounding the trans-spliced leader SL1 exon in the 5S rRNA gene spacer regions of Dirofilaria immitis, Brugia malayi, and B. pahangi were determined after PCR amplification, aligned with the genus Onchocerca for comparison, and used for the prediction of secondary structures. The nucleotide sequence of this region in B. pahangi was first shown in the present study. Hypothetical secondary structures of the spacer region suggested that the SL1 transcript is capable to form a stable stem-loop structure which may render transposition of the SL1 sequence to mRNA molecules. A homologous sequence to Sm-binding site was assigned on a bulge loop. No significant difference was observed in adult worms of D. immitis irrespective of sex or location. No difference was apparent between the two species in genus Brugia.
This study was conducted to evaluate protective efficiency of three different protocols for vaccination in canine heartworm infection. To evaluate the three protocols of immunization, dogs were separately immunized with living larvae; 1) immunization with gamma-attenuated infective larvae, 2) with 50 μg/kg ivermectin-abbreviation, and 3) with chemical abbreviation plus Freund's complete adjuvant (FCA). Each group was composed of two dogs. All dogs used for this study were subcutaneously challenged with 100 intact third-stage larvae (L3) various days after the last immunization, and the worms in the pulmonary arteries and the right ventricle of the heart were recovered 17 to 25 weeks post-infection. The numbers and the sexes of the worms were determined. A mean of 38 worms was burdened in the group immunized with irradiated L3, 36 worms in the chemically-abbreviated group, but 15.5 worms in the group with chemical abbreviation plus FCA. The percentages of the protection in the former two groups were nearly 50%, but 72.3% in the group with ivermectin plus FCA. The adjuvant enhanced the protective immunity against L3 challenge. Obvious eosinophilia was observed in both immunized and control dogs except for two dogs. There was no correlation between the suppression of eosinophilia and the protective immunity in the present study.
Serological titers to Pneumocystis carinii (Pc) and porcine reproductive and respiratory syndrome virus (PRRSV) were measured on a herd with epidemic Pc pneumonia (case herd) and two comparison herds, by an indirect fluorescent-antibody technique. In the case herd, the geometric mean titer (GMT) for Pc were 1:80 in pigs 1 week old, 1:10 in pigs 5 weeks old, and 1:80 to 1:190 in pigs over 6 weeks old. GMTs for PRRSV were >1:145 in most of age groups over 7 weeks old. In comparison herds, Pc and PRRSV antibody titers were low in weanling pigs. The results clarified the kinetics of antibodies to Pc and concurrent infection of PRRSV in the case herd.
A hepatoblastoma was found in a 13-year-old female Maltese dog. Histologically, the tumor showed a wide trabecular pattern and was frequently accompanied with vascular lake formation. Tumor cells were positive for cytokeratin and neuron specific enolase, but negative for chromogranin. Electronmicroscopically, tumor cells were accompanied with continuous basement membrane and had poorly developed desmosomes. Sinusoidal endothelia had fenestration and were surrounded by myofibroblast-like cells. To the best of our knowledge, this paper is the first report of morphological studies on canine hepatoblastoma.
The FOK rat was established as an inbred strain with genotypic adaptation to a hot environment. To investigate the mechanism of the heat resistance of FOK rats, heat production by isolated brown adipocytes in FOK and WKAH rats was measured at 37.0 and 39.9C. Basal heat production by brown adipocytes of FOK rats was not significantly different from that of WKAH rats. Isoproterenol increased the heat production in a dose-dependent manner in both strains, while 10-9 M isoproterenol caused a significant increase in WKAH rats and a negligible change in FOK rats. These results indicate that the reason why FOK rats are resistant to the severe heat is partly due to the low heat production by brown adipocytes.
Dose effect of inhibin antiserum on ovarian response and hormonal profiles were investigated. On day 12 of the estrous cycle (day 0=estrus), 14 of 19 cows were given a single i.v. injection of 25 ml (n=4), 37.5 ml (n=5) or 50 ml (n=5) antiserum against inhibin produced in a castrated male goat. The other 5 animals were given 50 ml castrated male goat serum (control serum). The animals in each group received a single i.m. injection of 0.5 mg prostaglandin F2α analogue (PG) 48 hr following the serum injection. The population of follicles and ovulation rate (estimated by the number of corpora lutea) were examined by ultrasonography. Administration of inhibin antiserum consistently resulted in a significant (p<0.01) increase in plasma concentrations of follicle-stimulating hormone (FSH) in inhibin-neutralized groups, although the increased FSH levels were sustained longer in 50-ml group than in the 25- and 37.5- ml groups. Levels in the circulating inhibin antibody titer were positively correlated with dosage of inhibin antiserum. A large number of antral follicles (≥ 4 mm in diameter) developed similarly after hypersecretion of FSH in all neutralized groups, coupled with a rise in plasma estradiol levels, while the number of large follicles (≥ 10 mm in diameter) on estrus showed a dose-dependent increase. Multiple ovulation (2 to 4) was recorded in all animals after injection of 50 ml inhibin antiserum, however all cows in the 25-ml group experienced only one ovulation and injection of 37.5 ml resulted in a variable number of ovulations (1 to 5). These results demonstrated that administration of inhibin antiserum on day 12, followed by injection of PG, was able to induce hypersecretion of FSH and subsequently multiple ovulations. The number of large follicles on estrus day and ovulations were affected by dosage of inhibin antiserum and were correlated with persistence of increased FSH levels or circulating antibody levels.
We devised a versatile intrathecal injection method enabling evaluation of central nervous system (CNS) toxicities in rats, and then explored appropriate physico-chemical properties for injectable test solutions at relatively large dosage volumes. In a preliminary bolus injection study, a 25-gauge needle was inserted into the subarachnoid space through the lateral aspect of the intervertebral portion between the 2nd and 3rd lumbar vertebra under light ether anesthesia. When 3% phthalocyanine blue solution, a vital dye, was injected in order to confirm its transfer to the brain tissue, the dye reached the cerebral ventricle, perivascular spaces and veins of the cerebral cortex. On the basis of these information, a fine polyethylene tube was introduced up to a level around the axis via a 20-gauge needle inserted in advance into the vertebral space by the same way. The needle was then withdrawn from the space leaving the tube filled with a test solution. After a full recovery from anesthesia, the infusion was commenced. Injection volume-versus-speed or osmolality-versus-pH relationship was assessed using solutions with various compositions, followed by monitoring mortality coupled with clinical signs. The tolerable combination of factors for the intrathecal solution without causing death was thought to be an injection volume up to 2 ml/rat with 300 mOsm/kg H2O, pH 3 to 7, at a injection speed of less than 0.5 ml/min, although minor clinical signs were observed. Pathological examination revealed pulmonary edema in dead animals, but no changes in surviving animals. This method applicable to conscious rats is considered to be simple and reliable, and does not require surgical operation and special equipment. The toxicological event in the intrathecal route seems to depend largely on the physico-chemical characteristics of the injectable solution. under these experimental conditions.
Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.
The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu)) was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2 strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an MW of approximately 47-51 and 39-44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2) (approximately 51-55 and 41-46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live vaccine.