To study the luteal and placental function of pinnipeds, we analyzed the localization of steroidogenic enzymes (P450scc, 3 β HSD and P450arom) in the corpus luteum and the placenta of ribbon seals (Phoca fasciata) and Steller sea lions (Eumetopias jubatus) immunohistochemically. P450scc and 3 β HSD were present in all luteal cells of both species. Almost all of the luteal cells were immunostained for P450arom, while P450scc and 3 β HSD were negatively immunostained in placentae and P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) corpora lutea of both species synthesize pregnenolone, progesterone and estrogen during the entire pregnancy period, and 2) like other terrestrial carnivores in the suborder Caniformia, placentae of both species do not have the capability for synthesizing progesterone in the latter half of active pregnancy period.
Localizations of carbonic anhydrase isoenzymes (CA I, CA II and CA III) were investigated immunohistochemically in the salivary glands and intestine of mature and suckling pigs. Carbonic anhydrase isoenzymes were not detected in the salivary glands of sucklings, but were present in the adult. Bicarbonate ion in saliva might be important for the digestion of solid foods in mature pigs, but unnecessary for the digestion of milk in sucklings. Expressions of CAI and CAII were detected strongly in the large intestine of the adult and sucklings, and faintly only at duodenum in the small intestine. CA I and CA II isoenzymes in the large intestine may be involved, at least in part, in ion absorption and water metabolism during digestion and absorption of milk in suckling pigs. In addition, CA I and CA II expression in the duodenal villus enterocyte may support the process of bicarbonate absorption in the duodenum.
Three optic nerves (L1, R2, R3) 12-18 mm behind the eyeball of the horse (Thoroughbred) were investigated quantitatively under light and electron microscopes. Thin sections at the thickness of 0.35 μm were cut, stained by toluidine blue and observed under the light microscope. The areas of the optic nerve and the axon bundles were 20.03 ± 1.04 and 16.59 ± 0.79 mm2 (mean ± SD, n=3), respectively. The axon numbers for optic nerve L1, R2 and R3, estimated from light micrographs, were about 481 × 10 3 , 543 × 103 , and 494 × 103 , respectively. Axons of optic nerve L1 were also counted from electron micrographs and the total number of 488 × 103 was received. Furthermore, axon diameters of optic nerve L1 were also measured from electron micrographs. The diameter of a circle with the same peripheral length as an axon, was regarded as its diameter. The medullary sheath of the axon was not included during measuring. Altogether 5,744 axons were measured and axon diameters were in a range of 0.23-12.69 μm, with a mean of 2.56 ± 1.45 μm (mean ± SD). A regional difference of axonal diameters was found across the optic nerve: the mean diameter of axons in the centro-dorsal region (2.28 μm) was the smallest, and had significant difference with those in several peripheral regions (P<0.05).
In May 1994, about fifty Japanese quails out of ninety being bred for experimental purposes at Miyazaki University died of acute septicemia within a few days. At autopsy, there were no gross pathological lesions, however, severe bacteremia was observed in all cases. Bacterial examination revealed the presence of Pasteurella multocida in blood and several organs in pure culture and they were of Carter's capsular type A, Heddleston's type 3-4 and Namioka's type O-8-9. The LD50 of bacteria in quails and mice were 4.3 × 104 cfu and 3.9 × 10 2 cfu, respectively. All of the three chickens experimentally infected with 4 × 104 of the isolate died within 20 hr after the infection and several bacteria were recovered from their blood and organs. This, to our knowledge, is the first report on an outbreak of fowl cholera in Japanese quails in Japan.
Excitatory amino acid transporters play important roles in termination of glutamatergic neurotransmission and protection of neurons from the excitotoxicity of glutamate in the central nervous system. We herein report isolation of cDNA clones of two distinct excitatory amino acid transporters, GLT-1 and EAAC1, from canine brain cortex by PCR-based cloning, and characterization of these transporter subtypes. Canine GLT-1 and EAAC1 exhibited Na+-dependent glutamate transport with high affinities in a Xenopus oocyte expression system. Despite the similarity in transport kinetics and in the predicted primary structures, GLT-1, EAAC1, and the previously identified GLAST showed different sensitivities to several structural analogues of L-glutamate. In addition, transcripts of these transporter subtypes showed distinct regional distribution in the brain in RT-PCR analysis, suggesting that excitatory amino acid transporters have distinct physiological and pathophysiological roles in the brain.
Concentrations of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-γ- and TNF-α-specific ELISA. In serum and whey samples from healthy cows, IFN-γ was almost undetectable and TNF-α was detected at low levels. At the onset of illness, INF-γ in sera and whey and TNF-α in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-γ and TNF-α in whey from mastitic cattle decreased significantly as the cows recovered.
CD34 is a leukocyte antigen that is expressed in various cell types including hematopoietic cells. Monoclonal antibodies against human, murine, and canine CD34 proteins have been used for the identification of lymphohematopoietic stem/progenitor cells. The cDNA encoding bovine CD34 was cloned, and its nucleotide sequence was determined. The identity of the deduced amino acid sequence of the encoded protein to those of human, murine, and canine CD34 proteins was 61.1%, 56.0%, and 66.1%, respectively. Northern blot hybridization with the cDNA as a probe detected CD34 RNA expression in the cerebrum, spleen, heart, and lung of a fetal calf.
ErbB subfamily genes, known as proto-oncogenes, encode receptor tyrosine kinases, and are expressed in relation to tumorigenesis of the mammary gland in humans. In this study, we examined the expression of erbB subfamily mRNAs in two canine normal mammary glands and 12 mammary tumor samples by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Each primer set was designed from the nucleotide sequence of the region conserved in erbB subfamily cDNA among other species. No erbB subfamily mRNAs were expressed in the normal mammary gland. In contrast, all of the subfamily mRNAs were expressed in a benign mammary tumor, and more than one type of the subfamily mRNA were observed in 11 malignant mammary tumors. The length of RT-PCR products were 380 bp for erbB1, 500 bp for erbB2, 644 bp for erbB3, and 416 bp for erbB4. These sequences were highly homologous to the cDNA sequences of other species. Therefore, these results suggest that the expression of erbB subfamily mRNAs in canine mammary tumors plays an important role in tumorigenesis of the mammary gland.
To clarify the relationship between tumor necrosis factor (TNF) and insulin resistance in dairy cows affected with fatty liver, naturally occurring cases were investigated. The affected cows were classified into following three groups according to histopathologic findings of the liver: mild fat droplet deposition (group 1; n=11), severe fat droplet deposition (group 2; n=10), and cloudy swelling (group 3; n=8). Serum TNF activities in Group 2 (8.67 ± 2.16 U/ml) and Group 3 (11.65 ± 1.92 U/ml) were significantly higher than that in Group 1 (3.57 ± 0.81 U/ml) (p<0.05). The insulin-tolerance tests showed that the insulin-stimulated glucose disposal rates (GDR) in Group 2 (27.6 ± 7.8%) and Group 3 (15.8 ± 9.1%) were significantly lower than that in Group 1 (41.7 ± 9.8%). There was a significant negative correlation between serum TNF activity and GDR in affected cows (r=-0.56, p<0.01). These results indicate that serum TNF activity is correlated with insulin resistance in cows with fatty liver.
Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.
A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 μl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.
The expression of inducible nitric oxide synthase (iNOS), an enzyme that produces nitric oxide, was examined in the hearts of pigs infected with porcine enterovirus serotype 3 (PEV3). Piglets orally infected with PEV3 developed tremors and paralysis 3-7 days post-infection. Affected animals had pericarditis and myocarditis. There were fibrin and inflammatory cell infiltrates (macrophages and neutrophils) in the pericardial sac and myocardium. Immunohistochemically, the majority of inflammatory cells in the pericardial sac were positive for iNOS and nitrotyrosine, an end product of nitric oxide. These results suggest that iNOS is upregulated in the pericardial lesion, and that increased nitric oxide production plays an important role in the development of PEV3-induced pericarditis and myocarditis.
Agents of transmissible spongiform encephalopathy (prion) are known to be extremely resistant to physicochemical inactivation procedures such as heat, radiation, chemical disinfectants such as detergents, alcohols, glutaraldehyde, formalin, and so on. Because of its remarkable resistance, it is difficult to inactivate prion. Chemical inactivation seems to be a practical method because it is applicable to large or fixed surfaces and complicated equipment. Here, three epoxides: β-propiolactone, propylene oxide, and glycidol (GLD) were examined of their inactivation ability against scrapie-mouse prion protein (PrPSc) under various conditions of chemical concentration, incubation time, and temperature. Among these chemicals, GLD worked most effectively and degraded PrP into small fragments. As a result of the bioassay, treatment with 3% GLD for 5 hr and 5% GLD for 2, 5 hr or 12 hr at room temperature prolonged the mean incubation time by 44, 30, 110 and 73 days, respectively. From dose-incubation time standard curve, the decrease in infectivity titers were estimated as 103 or more. Therefore, degradation of PrPSc by GLD decreased the scrapie infectivity. It is also suggested that pH and salt concentrations influence the effect of GLD. Although further study is necessary to determine the optimal condition, GLD may be a potential prion disinfectant.
Cecal contents (16 samples/each flock) of broilers derived from 212 flocks were investigated for colonization of Campylobacter from 1995 to 1999 in the southern part of Japan, and the isolates were tested for antimicrobial susceptibilities. C. jejuni-positive flocks numbered 42 (19.8%) and C. coli-positive ones 26 (12.3%); Campylobacter spp. were recovered from 68 flocks (32.1%) in total. MICs of ampicillin, erythromycin (EM), tetracycline, nalidixic acid (NA), norfloxacin (NFLX), and ofloxacin (OFLX) to these 68 Campylobacter isolates were determined. Quinolone-resistant Campylobacter isolates numbered 22 (32.4%). All the isolates except one were cross-resistant to NA, OFLX, and NFLX. A high frequency of quinolone-resistance was found in both C. jejuni and C. coli, whereas a high level of EM-resistance was found in only C. coli strains. All C. jejuni isolates were sensitive to EM.
A comparison between a bovine jugular vein treated with a hydrophilic polyepoxy compound cross-linker (Denacol), and expanded polytetrafluoroethylene (EPTFE), an artificial material, as a patch graft for the reconstruction of the right ventricular outflow tract under extracorporeal circulation in dogs, as if they had pulmonic stenosis, was made. Hemodynamic and histological examinations were conducted two weeks after the transplantation. Hemodynamic problems were not observed in either the Denacol or EPTFE groups. Macroscopically, organization of new tissue on the graft surface was more marked in the EPTFE group than in the Denacol group, and newly-formed tissue was seen surrounding the border of the graft and burying it in the EPTFE group. In the Denacol group, microscopic findings revealed the presence of inflammatory cells and fibroblasts, and an invasion of the graft by collagen fibers and elastic fibers. In the EPTFE group, there was minimal cellular infiltration of the graft and a thick layer consisting of collagen fibers and fibroblasts was observed around the graft. These results indicated that two weeks after transplantation the graft was better assimilated and organized with blood vasculature in the patch graft in the Denacol group than in the EPTFE group.
A new cell line (CoMS) was established from a 3-year-old male mongrel dog with mast cell tumor of the oral mucosa. CoMS cells grow in suspension with a doubling time of 27.0 ± 0.7 hr. The cytoplasmic granules were formalin-sensitive, showed diverse appearances in their ultrastructural findings and contained heparin proteoglycan and neutral protease chymase. Calcium ionophore A23187, substance P and concanavalin A caused significant histamine release from CoMS cells, while compound 48/80 failed to release histamine. This cell line will make an available source for studies on canine mast cell tumors.
Chondroitin sulfate (CS) isomers, 6-sulfate (CS6) and 4-sulfate (CS4), change their ratio to each other in cartilaginous tissues with aging. In this study, a quantitative measurement method of CS6 and CS4 was developed, using capillary electrophoresis (CE). Various buffer solutions, pH, and digestion times were studied, and the use of 0.1 M Tris-HCl at pH of 8.0 allowed the isolation of CS6 and CS4 from CS most efficiently when combined with chondrotinase ABC at a concentration of 1 mU/μg of the substrate during a 3 hr digestion period. Amounts of newly synthesized CS6 and CS4 in the intervertebral disk chondrocyte three-dimensional culture were quantified by this method after the proteoglycans were extracted by equilibrium density centrifugation.
The possibility of producing HanWoo (Bos taurus coreanae) calves from transferable bovine embryos, obtained by interbreed nuclear transfer using Holstein cytoplasts and surrogates, was investigated. As donor nuclei, HanWoo fetal fibroblasts were used. Cells were induced into quiescence by serum deprivation for 4-7 days before nuclear transfer. In vitro matured Holstein oocytes were enucleated, and single donor cell was placed into the perivitelline space of enucleated oocyte. After reconstruction, the embryos were fused, activated and cultured. On day 7, the embryos that developed to the blastocyst stages were transferred into Holstein recipient cows on day 6 to 7 of estrous cycle (estrus=0). The reconstructed embryos were successfully fused (58.8%; 47/80), cleaved (91.5%; 43/47), and developed to blastocysts (29.8%; 14/47). Eleven blastocysts were transferred into 5 Holstein recipient cows. Two recipients were pregnant, confirmed by ultrasonography at day 60 of gestation. But, one of them was opened between on day 80 to 100 of pregnancy, and the other had a stillbirth on day 255. The stillborn calf was physically normal, and we couldn't find any evidence of anomaly. These results show that cells derived from HanWoo somatic cell lines can be reprogrammed by interbreed nuclear transfer and develop subsequently in vivo as well as in vitro.
To determine the effect of maturational age of porcine oocytes on the induction of activation and development following nuclear transfer, we investigated maturation rate and efficacy of oocyte activation treatment with an electrical stimulus (ES) and cycloheximide (CHX) at various timing of maturation culture. Most oocytes developed to the metaphase II (MII) stage after 32 hr of maturation culture. Both in newly matured (32 and 36 hr of age) and MII-arrested (42 and 48 hr of age) oocytes, ES followed by exposure to CHX for 6 hr caused higher rates of pronuclear formation than ES alone. Effect of maturational age of oocytes on the development of parthenotes activated with ES and CHX was then examined. The highest percentage of parthenotes developed to blastocysts was obtained when ES was added at 42 hr of culture. Finally, newly matured (33 hr of age) and MII-arrested oocytes (44 hr of age) were enucleated, fused with serum-starved pig fetal fibroblasts and activated with ES and CHX around suitable timing (39 to 40 hr of age) or later (50 to 51 hr of age), respectively. The frequencies of cleavage (58.8%) and blastocyst formation (13.6%) of newly matured oocytes were significantly (P<0.05) higher than those of MII-arrested oocytes (38.9 and 1.8%, respectively). These results demonstrated that the development of porcine nuclear transfer embryos can be improved by using complete matured oocytes as cytoplasts and activation treatment with ES followed by CHX treatment.
From 1997 to 1999, 29 cases of disorders were detected in cattle and horses that had been fed ryegrass straw imported from the U.S.A. These animals showed symptoms resembling ryegrass staggers and the clinical signs disappeared after removal of the straw. Endophytic hyphae were detected in the seeds of all straw samples that were responsible for the clinical cases. Lolitrem B concentrations in the straw ranged between 972 and 3740 ppb. Ergovaline concentrations were between 355 and 1300 ppb. Even though the concentrations of lolitrem B were lower than the toxic threshold proposed by Oregon State University in better part of the cases, our observations suggest the possibility that lolitrem B lower than the proposed threshold can bring disorders to sensitive individuals.
The airborne transmission of Classical Swine Fever (CSF) virus to susceptible pigs, as well as the effect of vaccination with the CSF virus PAV-250 strain was investigated on this mode of transmission. Experiment I: four pigs were inoculated with the ALD CSFV strain (104.3 50% TCID) by the intramuscular route, and at the onset of fever, they were introduced into an enclosed chamber. At the end of the experiment surviving pigs were sedated, anesthetized and euthanatized. Experiment II: four pigs were previously vaccinated with the CSF virus PAV-250 strain, and at 14 days post-vaccination they were challenged with the CSF virus ALD strain. In both experiments, four susceptible pigs were exposed to infectious aerosols by placing them in a chamber connected by a duct to the adjacent pen containing the infected animals and were kept there for 86 hs. In Experiment I, pigs exposed to contaminated air died as a result of infection with CSF virus on days 14, 21 and 28 post-inhalation. These four pigs seroconverted from day 12 post-inhalation. CSF virus was isolated from these animals, and the fluorescent antibody test on tonsils was positive. In Experiment II, a vaccinated pig exposed to contaminated air did not seroconvert, nor was CSF virus isolated from lymphoid tissues. However, mild fluorescence in tonsil sections from these pigs was observed. In conclusion, CSF virus was shown to be transmitted by air at a distance of 1 m to susceptible pigs. Vaccination with the PAV-250 CSF virus strain protected the pigs from clinical disease under the same conditions.
Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.