We have constructed recombinants of bovine herpesvirus-1 (BHV-1) which express pseudorabies virus (PRV) gB, gC, gD or gE either individually or in combination. To test the protective immunity, mice were inoculated with these BHV-1 recombinants and challenged three weeks later with virulent PRV. A BHV-1 recombinant, BHV-1/TF7-1, which express PRV gC, gD, gE and gI but not PRV gB, protected all 7 mice from the challenge with 20 LD₅₀ virulent PRV and 6 out of 7 from the challenge with 100 LD₅₀ PRV, while one dead mice survived for 5 days after the challenge. All the control mice died in 3 days. BHV-1 recombinants which express PRV gB, gC and gD individually also gave some protection but not so effective as BHV/TF7-1. Before challenging with virulent PRV, sera were collected from immunized mice and antibody against PRV was assayed. Western blot analyses indicated that all the recombinants induced antibody in mice against PRV gB, gC or gD individually or in combination. Virus neutralizing (VN) titer against PRV was highest in mice which were inoculated with BHV-1/TF6-1, which expresses PRV gB. BHV-1/TF7-1, which was more effective to protect mice from the challenge of virulent PRV, induced lower VN titer.

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To clarify the biological behavior of karyomegalic alveolar epithelial cells induced by N-methyl-N-nitrosourethane (MNUR) and whether these cells progress to lung tumors, female Syrian golden hamsters, 6 weeks old, were given five subcutaneous injections of 0.6 mg/animal of MNUR at two week intervals and their lungs were examined at weeks 1, 4, 8 and 12 after the termination of treatment. At week 1, in severely affected areas where marked multifocal thickening of alveolar walls due to interstitial edema and cellular infiltration was observed, some regenerative alveolar epithelial cells had abundant eosinophilic cytoplasm and gigantic bizarre nuclei. The cells were confirmed ultrastructurally to be derived from alveolar type II cells. The number of these karyomegalic epithelial cells became significantly decreased thereafter, together with the reduction of inflammatory changes. On AgNOR staining, normal alveolar epithelial cells had 1.8±0.03 black dots within their nuclei while the karyomegalic epithelial cells had 4 black dots or more, from 1 week. The PCNA labeling index of the karyomegalic epithelial cells at week 1 was 14.6±2.4, and was significantly decreased from 4 week. This epithelial cell population also displayed a wider range of DNA contents (2.1-5.5C) than normal epithelial cells (1.6-2.3C). These results suggest that karyomegalic alveolar epithelial cells may be mutant cells which occur after initiation with MNUR, but the possibility that they can act as progenitors of alveolar epithelial cell tumors was considered to be extremely low.

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The composition of ciliates obtained from the forestomachs of eleven dromedary (one-humped) camels in Egypt was examined. As a result, eight genera containing 24 species with 11 forms were identified. Of them, one species was concluded to be new, then described as Dasytricha kabanii n. sp. This new species was clearly distinguished from D.ruminantium, the other species of the genus, by its lack of somatic cilia on the posterior one-fifth of the body surface. Entodinium nanellum and Epidinium ecaudatum f.caudatum were found in all camels examined. Although the percentage composition of respective species varied with the individual camel, the rate of Entodinium spp. was high in general. Total ciliate density in forestomach fluid was 1.9×10⁵/ml on average. Ciliate composition in Egyptian camels was similar to that in Bactrian camels, Camelus bactrianus, in China reported previously. However, more Entodinium species were detected from Egyptian camels than from Bactrian camels.

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From September 1993 to November 1995, to clarify the natural infection of Gnathostoma nipponicum (G.n.) larvae in small wild mammals, a total of 313 animals consisted of three species of rodents and two species of insectivores, collected from an endemic area of this nematode in Aomori Prefecture, were investigated. Two species, Rattus norvegicus (R.n.) and Chimarrogale himalayica (C.h.) were infected with G.n. larvae, and the infection rate was extremely high with 27.2% and 72.7% in the former and latter. The infected animals harbored the larvae of 2-10 (mean: 6.0) in R.n. and 2-40 (10.6) in C.h., and a total of 273 larvae was recovered. The all larvae (18) from R.n. and the vast majority of the larvae (216/255: 82.4%) from C.h. were recovered from the musculature, and many encapsulated larvae were found in these portions of C.h. Taxonomically, all the larvae were identified as the advanced third-stage larvae (AdL3) of G.n. The body length was 1, 056-2, 110 μm, and was larger than those of the naturally-infected AdL3 reported from cold-blooded animals. No morphological alterations or death of the larva was observed. From these results, it would seem that R.n. and C.h. are highly susceptible to G.n. larvae, and has very suitable characteristics as the hosts. Furthermore, a hypothesis is advanced that these mammals serves as the plausible paratenic hosts in the life cycle of G.n. This report is the first record of R.n. (Rodentia) and C.h. (Insectivora) naturally-infected with AdL3 of G.n.

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Salmonella Typhimurium (S.Typhimurium) originating from a naturally infected zebra finch (Poephila guttata), a common cage bird, was examined for the infectivity to the same species of the birds because of its importance to public health. Eight birds per group for each experiment were used. When zebra finches were inoculated orally with 10², 10⁴ or 10⁵ colony forming units (CFU) of S.Typhimurium and observed for 7 days, all the birds receiving 10⁵ CFU of S.Typhimurium were positive for the Salmonella in the liver or the intestines and necrotic foci in the liver were observed in 7 birds. When zebra finches were inoculated with 10⁵ CFU of S.Typhimurium and observed for 22 days, the S.Typhimurium was found in fecal samples throughout the experimental period and the maximum S.Typhimurium counts in feces were 4.0×10³ CFU per gram. S.Typhimurium was recovered from the liver, spleen and intestines in 2 birds and necrotic foci in the liver were also observed in 6 birds. The results indicate that S.Typhimurium originating from the zebra finch is pathogenic to these birds and the persistence of the pathogen lasts at least for 22 days.

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Thirteen Sika deer (Cervus nippon Temminck) showing ataxia on a stock farm in the north eastern district of China were examined histopathologically. The principal pathological changes were spongy vacuolation and myelin deficiency in the white matter of the spinal cord and brain stem, fibrosis and rupture of the elastic lamina of the spinal arterioles, and mesothelial hyperplasia in the spinal arachnoid. Other findings included defective formation of the elastic laminae of the aorta, and the blood vessels in the kidney and lung, hemosiderosis in the spleen and liver, and lymphocyte depletion in the systemic lymph nodes. Copper concentrations were low in the serum and liver. In the white matter of the spinal cord and brain stem, demyelination appeared to coexist with dysmyelination and secondary myelin breakdown. It was inferred that decreased activity of copper containing enzyme induced various lesions. The possible role of copper deficiency in the pathogenesis of the ataxic conditions is discussed.

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A simple method to quantitatively evaluate atherosclerosis in the rabbit coronary arteries by measuring macroscopic lesion areas (%) was attempted in the present study. Sixteen rabbits were fed a 0.5% cholesterol diet for 15 weeks and then 9 rabbits were sacrificed whereas the remaining 7 rabbits were maintained for further 9 weeks on a normal chow (at week 24). The left circumflex coronary arteries (LCX) were excised from the rabbit hearts under stereoscopic observation. The prepared arterial strips of LCX were 38.7±7.1 mm long and all of them reached the cardiac apex from the orifice. At week 15, the lesion area in LCX was negligible (3.2±0.4%) whereas the aortic lesions significantly developed (50.0±7.6%). At week 24, atherosclerotic lesions in both LCX and aortas increased to 32.8±9.2% and 85.9±5.6%, respectively. This is the first report that determined the luminal surface areas of atherosclerotic lesions in rabbit coronary arteries. This method may be more practical and useful for quantitative evaluation of coronary atherosclerosis in a large number of rabbits than histological observations of serial sections of rabbit hearts.

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The ultrastructural characteristics of cyst wall are a useful clue to the identification of Sarcocystis species. Among the eight species examined, S.cruzi, S.sp. 1 from sheep and S.hircicanis had the thin cyst wall with long, tapered hair-like villar protrusions. The protrusions arose from the dome-like bulges of cyst wall and ran parallel to the surface. No clear ramification was observed in the tip of protrusion of these three species. S.tenella, S.capracanis, S.miescheriana, S.fayeri, and S.sp. 2 from Japanese deer had the thick cyst wall. The villar protrusions of the former three species were palisade-like in shape, but those of S.tenella and S.capracanis were slightly thinner than those of S.miescheriana. The villar protrusions of S.fayeri and S.sp. 2 were finger-like, but those of the former species were shorter and thicker than those of the latter species. S.fayeri had many minute depressions on the surface of protrusion in a reticular pattern. Microtubules in the core of protrusion were seen in S.miescheriana, S.fayeri and S.sp. 2 but not in the other species. Microdepressions were observed on the surface of cyst wall among the protrusions in all the Sarcocystis species examined, but their function was not made clear.

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Since between 1989 and 1991, broiler, broiler breeder and layer chickens reared in three different prefectures of Japan, Hyogo, Ibaraki, and Miyazaki, were diagnosed clinically as having swollen head syndrome (SHS) these flocks were surveyed for antibody to turkey rhinotracheitis (TRT) virus using a serum neutralisation (SN) test. TRT-specific SN antibody was found in flocks of chickens in 2 out of the 3 prefectures. Thereafter, particular in the summers of both 1993 and 1994 outbreaks of SHS occurred in almost all areas of major chicken production in Japan. Almost chicken flocks affected by SHS possessed TRT SN antibody. No chicken sera collected between 1972 and 1988 possessed any SN antibody to TRT virus. It is suggested that in Japan, TRT virus is widely prevalent in areas of major poultry production.

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Hereditary cerebellar vermis defect rats (CVD), a new neurological mutant, developed disorganized cerebellar cortical tissues. The postnatal development of the cerebellum in the CVD was examined histologically and immunohistochemically. A main pathological change in the CVD was abnormal perivascular cell aggregations, beginning to be observed from postnatal day 5. Around postnatal day 14, perivascular cells increased in number and many of them showed vigorous bromodeoxyuridine incorporation activities, as seen in the normal external granule cells (EGCs). The perivascular cells and EGCs were strongly positive for low affinity nerve growth factor receptor. These immunohistochemical results revealed that abnormal perivascular cells were heterotopic EGCs. The perivascular cells led to dysplastic abnormalities of lamination and abnormal cell positioning in the CVD. These findings indicate that abnormal perivascular aggregations of EGCs play an important role in the pathogenesis for cerebellar cortical dysplasia of CVD.

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By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.

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The effects of intralesional injection of newly synthesized natural-type human tumor necrosis factor (nh-TNF) on experimental brain tumors in rats were investigated. The repeated injection of 5, 000 U of nh-TNF into the tumor resulted in the prolongation of the survival time of the rats. More than half of the nh-TNF treated tumors were red, and were characterized by histopathological features of marked congestion of tumor vessels. Fibrin formations were also found in the tumor vessels. These histological findings were not observed in the control tumors. Furthermore, coagulative necrosis was observed in the center of some reddish tumors. Leukocytes adhering to vascular endothelium and infiltration of the leukocytes were also observed in the tumors of nh-TNF treated rats. In the immunohistochemical examination, these infiltrated cells were primarily polymorphonuclear leukocytes and macrophages. Expression of intercellular adhesion molecule-1 (ICAM-1) increased on the tumor endothelial cells after the administration of nh-TNF. These results suggest that repeated injection of nh-TNF has a therapeutic effect on brain tumors through its extensive influences on tumor vasculature.

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Ultrasound-guided transvaginal follicular aspiration (UTA) was performed on 8 multiparous Japanese Black cows which had not been successful in a conventional superovulation program. These cows were actively immunized with a synthetic peptide replica of porcine inhibin αN1-26 (pINH) conjugated with rabbit serum albumin (RSA) using Freund's complete adjuvant. Booster injections of the peptide were given at 6, 10, 14 and 20 wk after the primary injection. Twelve follicular aspirations were performed between 10 and 33 wk after the primary injection. Cumulus-oocyte complexes (COCs) were aspirated from follicles ≥ 2 mm in diameter. The recovered COCs were classified into 5 categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers), Grade 4 (denuded) and Grade 5 (expanded and fluffy cumulus cell mass or degenerated ooplasm). The recovered COCs available for in vitro fertilization (IVF; Grades 1, 2, 3 and 4) were matured with granulosa cells (1×10⁶ cells/ml) for 20 to 23 hr, fertilized with frozen-thawed spermatozoa, and cultured in vitro. At day 7 after insemination, a portion of the embryos were transferred to Holstein heifers. There were great variations among donors in the number of recovered COCs and in the numbers of COCs in each grade, and also in the developmental ability of fertilized COCs. IVF procedures were carried out on 685 oocytes and produced 120 embryos (17.5%). The transfer of 36 fresh IVF embryos resulted in 20 pregnancies (55.6%) at 60 days after the transfer. This study has demonstrated first, that the number of COCs collected from individual inhibin-immunized cows by UTA greatly varies among donors. Second, recovered COCs vary greatly in the classification of the morphological categories of surrounding cumulus cell mass among donors and had different developmental abilities when they were cultured after fertilization in vitro. Third, the production of pregnancy in previously non-productive problem donors is possible using the UTA technique.

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The influence of oxygen concentrations in the gas atmosphere on the development of IVM/IVF bovine embryos was determined by culturing them in a microdrop of modified synthetic oviduct fluid medium supplemented with amino acids, insulin and PVA (mSOFai). After removing the cumulus cells at 18 hr post-insemination, presumptive zygotes were cultured in mSOFai for 104-106 hr under 5% CO₂ with various O₂ concentrations (2.5 to 20%). Reduced O₂ (5-10%) improved the development to the morula stage, and 5% O₂ gave the highest development. In the next experiment, morulae obtained after 102-104 hr of culture, were further cultured for 50 hr in mSOFai with 2 mM glucose under 5 and 20% O₂. An increase in the mean cell number in blastocysts, but not in the frequency of blastocysts, was observed under 5% O₂. In the third experiment, zygotes were cultured for 152-154 hr in mSOFai under 5 and 20% O₂, or cocultured with bovine oviduct epithelial cells in TCM199 + 10% FCS under 5% CO₂ in air. Percentage of blastocysts for mSOFai in 5% O₂ doubled to that for 20% O₂, and was similar to that for coculture. Moreover, mean cell number in the blastocysts for mSOFai in 5% O₂ was significantly higher than that for coculture. Results demonstrate that oxygen concentration critically affects embryonic development through zygotes to blastocysts, and suggest that around 5% O₂ is optimal. It also indicates that bovine zygotes can be cultured up to the blastocyst stage using a chemically defined medium with rates similar to those of a conventional coculture system.

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Short axon (SA) cells in the olfactory bulb are subdivided into six types after Golgi impregnation, although their functional significance is not fully elucidated. In the present study, we examined the golden hamster olfactory bulb by immunohistochemistry to localize neurotransmitters, neuron-specific marker, and nitric oxide synthase (NOS) in the SA cells. Enzyme histochemical staining was also performed to detect the activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, which is identified with NOS. In the main olfactory bulb (MOB), neuropeptide Y (NPY)-, NOS-, and NADPH-diaphorase-positive SA cells were detected in the glomerular layer (GL), vasoactive intestinal polypeptide (VIP)-positive SA cells in the external plexiform layer (EPL), and NPY-, somatostatin (SOM)-, protein gene product 9.5 (PGP 9.5)-, NOS-, and NADPH-diaphorase-positive SA cells in the granule cell layer (GCL). In the accessory olfactory bulb (AOB), VIP- and PGP 9.5-positive SA cells were detected in the mitral/tufted cell layer (MTL), and NPY-, SOM-, NOS-, and NADPH-diaphorase-positive SA cells in the GCL. The common presence of NPY-, SOM-, VIP-, PGP 9.5-, NOS-, and NADPH-diaphorase-positive SA cells in both the MOB and the AOB may suggest that respective types of cells with the same immunoreactivity play the same role no matter where these cells are located in the MOB or the AOB.

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Immunohistochemical and ultrastructural studies were performed on the sinusoidal lining cells of eight canine hepatocellular carcinomas. The sinusoidal endothelial cells of the tumors had a positive reaction for both Factor VIII-related antigen and peanut agglutinin, but did not bind with Ulex europaeus agglutinin-I. Desmin- and lysozyme-positive cells were present along the sinusoids and perisinusoidal spaces of the tumor tissues, respectively, but were fewer in number compared with those of normal canine liver. α-Smooth muscle actin-positive cells outlining the sinusoids were frequently observed. Electron microscopy revealed that basement membranes were often formed beneath the sinusoidal endothelial cells, with rare fenestration. Macrophages were present around or within the sinusoids and tended to increase in number relative to the degree of tumor differentiation. Myofibroblast-like cells with various morphological features, consistent with α-smooth muscle actin-positive cells, were frequently found in the perisinusoidal space. The present study indicates that the sinusoidal lining cells of canine hepatocellular carcinoma have some phenotypic characteristics.

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Detection of plasmid from leptospires and a comparison of the plasmid profiles between virulent and avirulent strains were performed to investigate whether leptospires contained plasmid(s) associated with virulence. Virulent strains of Leptospira interrogans serovars copenhageni, lai, canicola and pomona, which were virulent for the guinea pig and/or hamster and which showed chemotaxis toward hemoglobin, contained approximately 370 kilobases (kb) plasmid. Avirulent strains of L.interrogans also contained identical plasmid. Similar plasmid profiles in virulent and avirulent strains of L.interrogans were observed. These data showed that no plasmids associated with virulence or chemotaxis were detected. Strains of saprophytic leptospires, L.biflexa and Leptonema illini, did not possess any plasmid.

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Antibody against Mycoplasma bovis in sera of 48 calves introduced to a farm, in which calf pneumonia associated with M.bovis had been occurring in the last 3 years, was detected by an indirect hemagglutination test. Significant rises of antibody titers in sera of calves belonging to the groups A (16 calves) and B (14 calves) were recorded by day 60 post-introduction. On the other hand, a significant increase of antibody titers of 18 calves in group C, which had been administered antibiotics as a preventive therapy, was demonstrated at day 248 after arrival. These results indicated that the spread of M.bovis infection occurred easily on the contaminated farm, and a preventive therapy could delay the outbreak of calf pneumonia associated with M.bovis.

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Thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus increased in cell number in the alimentary tract of a gastropod, Clithon retropictus, from an undetectable level in July to 2.0×10³/g in September 1991 at the Sada estuary in the southern part of Japan. It was not detected in microalgae, foods of the gastropod, at the estuary or muddy sediments at a neighboring fishing port. The strain was not detected at two estuaries and two fishing ports ecologically separated from the Sada estuary. Serotypes of the isolated strains were different from those registered as clinical isolates. Our results confirm that TDH-producing V.parahaemolyticus grows selectively in the gastropod at some estuaries in Japan.

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The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.

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Two cases of hypertrophic cardiomyopathy in Holstein dairy cows are presented. At necropsy, the hearts revealed proportionate hypertrophy of the entire ventricles. The cut surface showed relatively large areas of myocardial scarring scattered throughout the ventricular walls including the septum. Microscopic examination revealed marked disorganization of cardiac muscle cells, intramural coronary arteries with thickened walls and narrowed lumina, and pronounced myocardial fibrosis. These features resemble those of hypertrophic cardiomyopathy in humans, suggesting the presence of a similar primary myocardial disease in cattle.

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In two blacktailed prairie dogs (Cynomys ludovicianus), hepatocellular carcinoma with metastasis to the lung, co-existing chronic active hepatitis, and hyperplasia of hepatocytes in the non-neoplastic areas of the liver tissue were observed. The hyperplastic hepatocytes had many eosinophilic inclusion bodies positive for orcein stain in their cytoplasm. Electron microscopy revealed areas of a specific mail-like mesh structure in the location of the cytoplasmic inclusion bodies. Ultrastructure of inclusion bodies differed from that of the hepadna virus usually associated with hepatocellular carcinoma, and hepadna virus-like particles were not observed.

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