The immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin were observed in the tooth buds of fetuses of minke whale, Balaenoptera acutorostrata. Distributions of extracellular matrices (ECMs) examined in this study except for tenascin were generally similar to those of terrestrial mammalian species during development of the tooth bud. Tenascin in the fetuses of minke whale showed characteristic distributions in the dental lamina and the enamel organ in the early tooth developmental stage. In the physiological degeneration stage of tooth bud development, immunoreactivity of the ECMs were very weakly and limitedly detected in the dental papilla and the surrounding mesenchyme. Immunoreactivity of tenascin and type I and III collagens were positively detected in the developing baleen plate germ which was associated with the degenerating tooth bud. These findings suggested that expressions of the ECMs were related to the formation of the tooth bud and baleen plate germ, and that the lack of the ECMs was related to the degeneration of the tooth bud in the fetal minke whale.
The initial disorders of the epiphyseal growth plate cartilage were immunohistochemically examined in the proximal tibia of rats administered a high dose of vitamin A. Male Wistar rats were given 100, 000 IU/100 g body weight/day of vitamin A for administration periods of 1 to 5 days (Day 1 to 5) from 4 weeks after birth or were given deionized water and used as control. They were sacrificed after 5-bromo-2'-deoxyuridine (BrdU) injection on Day 1 to Day 5 to remove the tibiae. The tibiae were processed for immunohistochemical examinations using antibodies against type I, II, X collagens and BrdU. BrdU-incorporated chondrocytes and type X collagen-negative area were reduced since Day 2 and type X collagen-positive area was reduced since Day 4. The cartilage matrix partially lost type II collagen and deposited type I collagen in the epiphyseal growth plate near the periosteum on Day 5. These findings suggest that a high dose of vitamin A initially disturbed the differentiation from resting to proliferating chondrocytes, subsequently inhibited the differentiation from proliferating to hypertrophic chondrocytes, caused the chondrocytes to deviate from the process of normal differentiation, and finally resulted in the deformation of the epiphyseal growth plate.
Temporal and spatial distributions of cytokeratin (CK) polypeptides were detected by monoclonal antibodies (mAbs) K8.13 and K8.12 during the development of the bovine ruminal epithelium. By the Western blotting analysis after the sodium dodecyl sulfate-polyacrilamide gel electrophoresis, mAb K8.13 confirmed 60.8 and 63.0 kD CK polypeptides in the fetal ruminal epithelial extract, and mAb K8.12 also 48.0 and 54.0 kD CK polypeptides. Immunohistochemical reactivities against both mAbs were detected only in the epithelial cells throughout the fetal periods. Distributions of CK polypeptides detected only by mAb K8.13 were observed on the basal side of the epitherial layer, but not by mAb K8.12 in the 7 cm fetus in crown-rump length. MAb K8.13 reacted also intensely with columnar-shaped cells in the basal layer in the fetuses of the later developmental periods. These results suggest that CK polypeptides detected by mAb K8.13 might be involved in the differentation and/or the maintenance of the basal layer in the ruminal epithelial development.
Nerve endings showing calretinin immunoreactivity were examined in the lower respiratory tract of the adult rat. Tree-like nerve endings were immunostained in the tracheal and bronchial smooth muscle layer. The endings that arose from thick nerve fibers and formed corpuscles composed of many arborized nerve terminals. A few of the nerve endings were also observed in the lamina propria of the tracheal mucosa, close to the epithelial layer. Immunoelectron microscopy revealed that the immunoreactive terminals were filled with mitochondria and scattered among the intermuscular collagen fibrils. Schwann cell sheath and collagen fibrils were intercalated between the smooth muscle cells and nerve endings. The calretinin immunoreactive nerve endings observed in the present study seem to be slowly adapting stretch receptors.
Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66 % and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence.
The effects of atmospheric ammonia, a major pollutant in animal confinement facilities, on bovine neutrophils and bronchoalveolar macrophages were evaluated in vitro. Ammonia exposure at concentrations 50, 100 and 200 ppm for one hour impaired recovery rates of neutrophils dose-dependently but enhanced their chemiluminescence activity per cell at lower concentrations (50 and 100 ppm). Macrophages were resistant to the exposure. Their recovery rates and chemiluminescence remained unaffected even at 200 ppm exposure. The present results suggest that ammonia exposure is unfavorable for bovine neutrophils in vitro, and probably in vivo also, in light of causing cell damage and triggering wider inflammatory responses.
The effects of four antigenic fractions of Pasteurella multocida serotype A isolated from a duck in the Philippines on the phagocytic activity of chicken peripheral blood leukocytes were studied by a flow cytometer. These fractions were the lipopolysaccharide-protein complex (LPS), crude capsular antigen (CCA), ribosomal fraction (RS) and outer cell layer (OCL). Among these four antigens, only CCA but not LPS RS and OCL, significantly increased the phagocytic activities of mononuclear cells (MNC) and polymorphonuclear cells (PMN). This result indicates that CCA has an immunological property enhancing the phagocytic activities of MNC and PMN.
Visualization of copper-induced hepatitis (CuH) in LEC rats was performed by using an MRI apparatus equipped with a magnet producing a high magnetic field of 7.05 T. When three groups of LEC rats (6-16 [pre-hepatitis], 15-26 [acute hepatitis] and 40-77 [chronic hepatitis] weeks old) were examined by MRI under T2-weighted imaging conditions which are suitable for the diagnosis of human hepatitis, hypointense MR images of the livers were, as a whole, obtained in all groups, suggesting that these conditions were not adequate for imaging of CuH of LEC rats. The shortening of the T1 and T2 relaxation times of livers due to an excess amount of paramagnetic irons under the high magnetic field was responsible for the lowering of MR signal intensities of the livers, especially those of 15 to 26-week old rats showing acute hepatitis. However, theoretical calculation of the MR signal intensities using the T1 and T2 relaxation times of the livers indicated that their imaging might be possible under proton density-weighted conditions even with a high magnetic field. Experimental results showed that hepatic injury was visualized as hyperintense regions in the MR image of the liver in the acute-phase rat.
A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 × 105 independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot analysis. In situ hybridization revealed that the expression of follistatin mRNA in the equine follicle was restricted exclusively to granulosa cells. When the expression pattern of follistatin mRNA in the equine uteroplacental tissues from mid- to late-pregnancy was examined, it was shown that its expression level tended to decrease after mid-pregnancy. These results suggest that follistatin acts in the reproductive tissues of the mare in maintaining pregnancy.
Following stimulation by the transforming growth factor-β (TGF-β) family in the cytoplasm, the Smad family is phosphorylated and translocated to the nucleus and activates several gene transcriptions. In this study, the mouse Smad3 cDNA including the open reading frame (ORF) was cloned from the mouse brain using a RACE (rapid amplification of cDNA ends) technique, and its expression pattern was analyzed in mouse tissue using northern blot. The predicted amino acid (aa) sequences of mouse Smad3 showed a high homology with human Smad3 (99.3%) and mouse Smad2 (85.4%). It revealed that this protein may be highly conserved in different species of mammals. Northern blot analyses revealed that Smad3 was highly expressed in the brain and ovary, and that the size of major transcript was about 5.7 kb. In situ hybridization analyses revealed the high expression of Smad3 was detected in the pyramidal cells of the hippocampus, the granule cells of the dentate gyrus, the granular cells of the cerebral cortex and the granulosa cells of the ovary. Smad3 may be essential transducer of signals from TGF-β and activin in these cells.
The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization.
A cDNA encoding cysteine proteinase of Theileria sergenti was isolated from a piroplasm cDNA library and its nucleotide sequence was determined. The gene encodes a polypeptide of 402 amino acids with predicted molecular mass of 46.4 kDa. Analysis of the predicted amino acid sequence revealed a number of features common to known cysteine proteinases. Southern blot analysis showed that the cysteine proteinase gene was likely to be a single copy per genome.
A 32 kilodalton major piroplasm surface protein (MPSP) is expressed abundantly on the surface of intraerythrocytic piroplasms of Theileria sergenti and is considered to be a candidate antigen for vaccine development against piroplasmosis. In this study, transcripts of MPSP gene were detected in an expression cDNA library prepared from T. sergenti-infected tick salivary glands. Expression of MPSP in the sporozoite stage was also confirmed by immunoblot analysis. Its expression at the sporozoite and intraerythrocytic stages gives scope for possible induction of protective immunity being targeted at both stages by immunization with recombinant MPSP.
A field trial was conducted to evaluate the efficacy of a topical formulation of ivermectin administered at the dose of 500 γg/kg against horn flies (Haematobia irritans) in cattle. Eighty-eight cattle in four herds naturally exposed to horn flies were used in the trial. Replicates were formed of two herds. Within replicates, one herd was randomly allocated to the untreated control and the other to the ivermectin treatment group. Horn fly counts were taken on the treatment day (Day 0) and on Days 7, 14, 21, 28, and 35 post-treatment. There were no horn flies on any cattle in the treatment group, whereas all the control cattle were continuously infested by horn flies on each examination day.
Rumen ciliate species and composition were surveyed on the native sheep, Friesian-cattle and dromedary (one-humped) camels kept in Libya. As a result of survey, 5 genera including 14 species with 5 formae in native sheep, 9 genera including 27 species with 6 formae in Friesian-cattle and 6 genera including 13 species and 7 formae in dromedary camels were identified. All of the ciliate species and their percentage composition detected from the Libyan sheep and cattle in this examination were similar to those found from corresponding animals in the other countries. Libyan camels lacked some peculiar ciliate species found from camels in the other countries, but had many cosmopolitan species common with those in the domestic ruminants, suggesting that ciliate faunae of camel are easily affected by the other domestic ruminants kept together. The ciliate density was estimated as 105/ml in every host species.
Sarcocystis hominis was first isolated from slaughtered cattle raised in Japan. Cysts were 1, 220-4, 460 × 80-384 γm in size and their wall was 3 to 6 γm thick and appeared radially striated in the histopathological sections because of the presence of palisade-like villar protrusions on the surface. The protrusions were 3.1-4.3 × 0.7-1.1 μm in size and had many microtubules in the core. Two cynomolgus monkeys, Macaca fascicularis, fed with the Sarcocystis cysts began to pass sporocysts, which measured a size of 14.3-15 × 9.5-10 γm, in the feces 10 days after ingestion.
Five Thoroughbreds were classified into 4 groups according to the administration method used for saline solution (saline), ambroxol, and cephalothin sodium (cephalothin). In group A, cephalothin was injected intravenously after oral administration of ambroxol. In group B, cephalothin was injected intravenously after oral administration of saline. Groups C and D were used as control groups. The dose of cephalothin or ambroxol was clinically administrated. Venous blood and bronchoalveolar lavage fluid (BALF) were sampled from each group. In groups A and B, cephalothin concentrations in plasma reached their maximum level 5 min after cephalothin administration and then declined over time. In plasma obtained from groups A and B, there were no significant differences in pharmacokinetic parameters (T½, Kel, Vd). By contrast, cephalothin concentrations in BALF reached their peak at 180 min after cephalothin administration in both groups A and B and maintained a relatively high level even after 300 min. These findings indicate that cephalothin requires a relatively long period of time to move from the blood stream to the alveolar cavity, but once transferred to the alveolar cavity, it is preserved for a long time. In groups A and B, cephalothin concentrations in BALF were approximately at the same level. However, in group A, total protein in BALF was lower at 60, 180, and 300 min than the other groups. Then, cephalothin concentration was adjusted to total protein in BALF. After adjustment to total protein in BALF, group A showed a concentration level of cephalothin approximately 1.5-fold higher than that of group B. This suggests that the transferability of cephalothin to the alveolar cavity improves as a result of the oral administration of ambroxol.
Cecal contents of 2, 345 broiler chickens consisting of 28 flocks originated from 12 farms were examined for the prevalence of Salmonella to know the actual status of infection with Salmonella in the chicken flocks. Salmonella was isolated from 336 (14.3%) samples. From these isolates, eight serovars were identified. Of the 336 Salmonella isolates, 242 (72.0%) were serotyped as S. Blockley, 60 (17.9%) S. Hadar, 15 (4.5%) S. Bredeney, nine (2.7%) S. Schwarzengrund, four (1.2%) S. Anatum, three (0.9%) S. Enteritidis, two (0.6%) S. Ohio, and one (0.3%) S. Livingstone. The same serovars of Salmonella were repeatedly found in the chickens from the same farms. S. Typhimurium and S. Enteritidis were detected in pooled broken eggshell samples collected from the hatchery. Analysis of plasmid profiles revealed 11 patterns of S. Blockley and seven patterns of S. Hadar. Strains of the same plasmid profiles of S. Blockley were isolated repeatedly from the same farm over one year after the first isolation.
A mixture of bacteria, having a methane-utilizing ability, was separated from a bioreactor supplied with air and methane gas. The bioreactor was operated to treat trichloroethylene (TCE)-contaminated groundwater. The mixture was composed of an obligate methane-utilizing bacterium and a heterotroph, identified as Methylomonas methanica and Pseudomonas sp., respectively. The mixed culture of these two strains removed TCE. In addition, it appeared that a cooperative metabolic interaction of these strains enabled Meth.methanica to maintain the TCE degradation ability.
Occlusion of the internal carotid artery by insertion of intravascular platinum microcoils for guttural pouch mycosis was experimentally evaluated in 9 healthy adult Thoroughbred horses. The internal carotid artery was ligated to its origin, and an arteriotomy was made distal to the ligature, which was then occluded by insertion of the microcoil approximately 13 cm distal to its origin. Cessation of blood flow was determined visually and by angiography at the arteriotomy site. Six horses were evaluated for complication clinically and by endoscopy after surgery. One horse was necropsied after 30 days of surgery for histological evaluation of artery thrombus formation. In the other 3 horses, the blood flow of the right internal carotid artery was monitored, before and after microcoil occlusion of the left internal carotid artery. One or 2 microcoils stopped blood flow within a few minutes. No other abnormal findings were observed clinically. Thrombus was observed in the occluded segment of 1 horse 30 days after insertion; but no abnormalities were detected. The blood flow in the right internal carotid artery increased by approximately 28-58% after occlusion of the left internal carotid artery. This microcoil vascular occlusion technique causes an effective thrombosis, and based on experimental studies and clinical application in 2 horses with epistaxis due to guttural pouch mycosis, this technique would appear to be safe and efficacious.
For objective evaluation in the lung arterial lesions, density histogram revealed by survey thoracic radiographies of fifteen canine filariosis and five normal canine were digitally analyzed, and preparation of pulmonary artery angiogram with inflated-fixed lung, the changes in the histogram and the pulmonary arterial lesion by a soft x-ray examination were compared. In the lung areas affected by filariosis, the density histogram increased the white level and decreased the black level in each part compared to a normal lung. In comparison with the normal parameters, those of the filariosis it were significantly increased in minimum grey level values (Min), maximum grey level values (Max), and the maximum frequency grey level values (Mode) and, it was significantly decreased in maximum frequency values (MaF). The pulmonary arterial lesion of the filariosis showed obvious morphological changes such as in distinction, pruning, angiectasis, and meandering. In the grade of pulmonary arterial lesion, the parameter Min, Max, Mode and MaF were changed significantly. From these results, it was clear that the methods for the lung arterial lesions analysis of x-ray images were confirmed to be highly beneficial in the lung arterial lesions for objective diagnosis.
Four types of commercially available feline calicivirus (FCV) vaccine were compared in terms of their efficacy on the basis of the ability of the sera of specific-pathogen-free cats immunized by two injections of each type of vaccine to neutralize FCV field isolates. Each vaccine immune serum neutralized relatively well strains F4, F9, and 255, which were FCV laboratory strains. As to 36 strains of field isolates, however, vaccines A, B, C, and D immune sera did not neutralize 18-20 of the strains (50.0%-55.6%), 19-22 of the strains (52.8%-61.1%), 22-25 of the strains (61.1%-69.4%), and 8-16 of the strains (22.2%-44.4%), respectively. These results indicate that there is much difference in neutralizing antigenicity between the existing vaccine strains and the FCV strains that are prevalent in Japan, suggesting the need for improvement of FCV vaccines.