Familial amyloidotic polyneuropathy (FAP) is a hereditary disease characterized by the systemic accumulation of amyloid fibrils. A mutant transthyretin (TTR) gene is mainly responsible for the disease. However, the variable age of onset and low penetrance might be due to environmental factors, one of which is the intestinal flora. Three types of intestinal flora were introduced into a transgenic (Tg) mouse FAP model, 6.0-hMet30. The CV1 and CV2 group transgenic mice were transferred with the intestinal flora from two different mouse facilities housed under conventional conditions, and the SPF group transgenic mice were kept under specific pathogen free conditions in our facility. All the mice were maintained under controlled temperature, humidity and bacterial conditions. Over a period of 28 months, amyloid was not deposited in the SPF and CV1 groups. In contrast, amyloid was deposited in the esophagus and small intestine of two of the three CV2 mice at 18 months. Many neutrophils infiltrated the lesions. The numbers of tissue neutrophils were higher in the CV2 group than in the SPF and CV1 groups at 18 months. The CV2 flora included fewer gram-positive anaerobic cocci as well as higher proportions of yeasts, staphylococci and enterobacteriaceae compared with the SPF and CV1 flora. These findings suggest that the intestinal flora plays an important role in amyloid deposition.

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Mice and rats are frequently subjected to long-term raising in studies of aging. These animals are usually given growing or breeding diets from a young age. This raising method causes diseases such as chronic nephropathy with proteinuria due to nutritional excess. Consequently, a long-term raising study on male F344/DuCrj rats using nine sorts of diets differing in crude protein (CP; 12, 28, 44%) and digestible energy (DE; 2.8, 3.7, 4.5kcal/g) contents was carried out. It was found that feed consumption was regulated by DE, not digestible crude protein (DCP) intake. Body weight was controlled within low energy areas, and was not influenced by feed or DCP intake. The liver and kidney weight at 105 weeks of age increased in response to an increase in the level of CP in the diet. Chronic nephropathy was severe in rats fed high protein diets and moderate levels of protein with moderate to high energy diets. Fatty liver and bile duct hyperplasia were found in rats fed a high protein and high energy diet. Few pathological findings of kidney and liver were found in the low protein and low energy diet group. The reduction of disorders attributable to excess energy or inappropriate diet suggests that low protein and low energy diets are most suitable for long-term raising in this strain of rat.

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Electron beam sterilization for laboratory animal diets was examined as an alternative to ⁶⁰Co gamma rays. Solid, powder diets for “mice and rats” and solid diets for “rabbits and guinea pigs” which are the main products sterilized by ⁶⁰Co gamma rays were irradiated with 10-MeV electrons from a linear accelerator at the Research Institute for Advanced Science and Technology, Osaka Prefecture University. At least 20 kGy was required to sterilize the samples irrespective of solid or powder diets, which was in good accordance with the results for ⁶⁰Co gamma rays. Using a set dose of 30 kGy, a thickness of 45 mm for solid diets and 30 mm for powder diets could be sterilized by “one-sided” irradiation. “Dual-sided” irradiation could sterilize all the solid diets and the powder diets contained in the thicknesses of 90 mm and 75 mm, respectively. Irradiation effects of 10-MeV electrons on the nutrient quality of each diet were almost equivalent to those of ⁶⁰Co gamma rays. These results suggest that commercially adopted sterilization doses for ⁶⁰Co gamma rays are applicable to electron sterilization without modification if the depth-dose profile and the minimum dose of irradiated samples are precisely assessed.

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A cDNA coding Leishmania homologue of receptors for activated C kinase (LACK), which was known to play an important role in the early phase of Leishmania infection, was molecularly cloned from Leishmania amazonensis promastigote by using reverse transcription and nested polymerase chain reaction, and was sequenced. The L. amazonenis LACK cDNA showed 97.3 to 99.3% homology and its deduced amino acid sequence showed 98.7 to 99.7% identity in comparison with LACK sequences from five other species. The amino acid sequences in the immunodominant peptide region were completely conserved among Leishmania spp. tested. Intravenous pretreatment of the recombinant L. amazonensis LACK into BALB/c mice showed progressive lesion development compared to PBS (−) injected control mice, suggesting the important role of LACK in the early phase of L. amazonensis infection.

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This paper reports the results of reproduction with 45 wild African green monkeys (Cercopithecus aethiops) (36 females and 9 males) during the nine years from 1981 to 1989 under indoor individually-caged conditions. In 206 cases of menstruation observed, menstrual discharge lasted for 2.5 ± 1.2 days in cycles of 22-48 days, and the length of each menstrual cycle was 31.2 ± 6.5 days. Females who had regular menstrual cycles were subjected to “one-to-one timed mating”; females and males were put together on a one-to-one basis daily only for a certain period of time on and after the day of ovulation. Females who had irregular menstrual cycles or had no menstruation were subjected to “every-other-day mating”; females and males were put together on a one-to-one basis every other day for at least 16 weeks. The pregnancy rate (No. of pregnant females/No. of mated females) by one-to-one timed mating was 48.9% (116/237); 2.0 mating trials were needed to obtain one case of pregnancy. On the other hand, the pregnancy rate (No. of pregnant females/No. of mating trials) by every-other-day mating was 96% (48/50). Females who delivered normally totaled 129. The mean gestation period was 165 days when males, weighing 343 g on average at birth, were delivered, and 166 days when females, weighing 318 g on average at birth, were delivered. The male and female newborns were nursed for 131 and 138 days, respectively, on average. Details are summarized in Table 3. This paper also reports 23 cases of abortion, 6 stillbirths, and 6 cases of Caesarean section, by which three live fetuses and three dead fetuses were obtained.

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We studied characteristics of the daily pattern of heart rate (HR), blood pressure (BP), body temperature (BT), and locomotor activity (LA) in conscious and unrestrained Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits and age-matched normal Japanese white (JW) rabbits, using a telemetry system. In all JW rabbits, nocturnal patterns were observed in HR, BT and LA. In the 5 months group of KHC rabbits, however, diurnal rhythm was observed in HR, and in the 10 months group of KHC rabbits, it was also shown in LA. The nocturnal pattern was observed only in BT in 10 months KHC rabbits. Mean blood pressure (MBP) in JW and KHC rabbits showed no clear daily pattern. The mean daily values of HR and BT were not altered between the 5 months and 10 months groups in KHC rabbits, although those in JW were lower in the 10 months group than in the 5 months group. Moreover, the daily values of HR and MBP in KHC rabbits tended to be higher than those in the age-matched JW rabbits. The pulse pressure in the 10 months group of KHC rabbits tended to be greater than the 5 months groups of KHC and JW rabbits. Furthermore, short-term variabilities in BP in the 5 months KHC rabbits were significantly lower than those in the other groups. From these results, it is suggested that the cardiovascular function, including the autonomic nervous function is altered with the development of atherosclerosis in KHC rabbits.

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The aims of this study were to investigate transplacental transport of α2-macroglobulin (α2M) in rats and to examine the degree of α2M induction in maternal and neonatal rats with acute inflammation. Serum was collected from healthy pregnant CD (IGS) rats, neonates of the pregnant rats and their cord blood. Additional serum samples were obtained from pregnant rats inoculated with an inflammatory agent, turpentine oil, their neonates and cord blood, and neonates inoculated with turpentine oil. The serum levels of α2M were measured by means of an enzyme-linked immunosorbent assay. The average serum levels of α2M in healthy neonates and cord blood were about 380 μg/ml. Serum a2M level in neonates inoculated with turpentine oil averaged about 580 μg/ml. Serum α2M levels in maternal rats inoculated with turpentine oil, neonates from those rats and their cord blood were elevated, the values being 2, 000 μg/ml or higher. It was demonstrated that induction of α2M in neonatal rats was lower than in maternal rats when inoculated with turpentine oil. These results suggest that α2M is transplacentally transported from maternal rats to fetal ones.

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A spray administration of ivermectin was evaluated for the treatment of pinworm infection in mice. In this study, a spray of 0.1% ivermectin injectable solution over the entire cage once a week, for three consecutive weeks (one cycle treatment), was effective in eradicating both Syphacia obvelata and Aspiculuris tetraptera from mice under experimental conditions. In addition, no acute toxicity was observed in 105 mothers or 687 neonates treated with ivermectin, indicating that ivermectin does not affect murine reproduction. Finally, we attempted to eradicate pinworms from infected mice in our institute using this method. Two cycles of treatment were administered, with a two-week pause between cycles, resulting in complete eradication for at least one year. Treating mouse colonies with spray ivermectin is inexpensive, safe, requires very little labor and is very effective at eradicating pinworms from mice.

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In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP^-/+ mice were composed of 9 OGP^-/-, 18 OGP^-/+, and 10 OGP^+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP^-/-, 12 OGP^-/+, and 4 OGP^+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.

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Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32°C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.

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An allele specific polymerase chain reaction with confronting two-pair primers (PCR-CTPP) was developed as an assay for genotyping the mouse Prkdc^scid gene mutation (former name scid). The reverse primer (WR) was designed to include the antisense nucleotide (A) specific for the wild type allele at the 3’ end with the counterpart forward primer (F) upstream. The other forward primer (MF) was designed to include the sense nucleotide (A) specific for the Prkdc^scid mutation at the 3’ end with the other counterpart reverse primer (R) downstream. PCR was performed in a single tube with these two pairs of primers. The products specific for each allele extended by F/WR (101 bp) or MF/R (180 bp) were visualized with common PCR products (257 bp) extended by F/R, and three genotypes of mice (Prkdc^scid/Prkdc^scid, Prkdc^scid/+, and +/+) were clearly distinguished.

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Although whey acidic protein (WAP) has been suggested to have some biological functions, its true function has not yet been clearly elucidated. We have generated transgenic mice ubiquitously and highly expressing the WAP gene. The pups born from one female among these transgenic mice showed low growth or died during nursing. This transgenic founder showed novel development of the mammary glands, and demonstrated normal parturition and nursing behavior. The mammary glands showed low-distended ductal structures, and poor development of lobulo-alveolar and acinous formations despite normal nursing, while mammary ducts were rather large in comparison with those of normal lactating females. Although this founder was found to be mosaic for transgenesis, it was shown to be a useful animal model for investigating WAP function.

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Enrofloxacin, a fluroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment.Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.

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Pseudorabies virus (PrV) YS-81 strain latently infected in 6-week-old BALB/c mice was detectable by nasal swabbing, and serum antibody was shown to increase in titer after intraperitoneal injection for 3 days with acetylcholine or dexamethasone.

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Tree shrews possess an intestinal diverticulum. We investigated this diverticulum with histological and immunohistochemical methods to determine whether this diverticulum was cecum or not. The ratio of the length of diverticulum/small intestine was apparently shorter than that of several primates. In the histological study, mucous membrane of the small intestine was shifted to that of the large intestine at the junction of the diverticulum. Histological features of the diverticulum were similar to those of the large intestine, but the shape of mucousal surface was rather simpler than that of the large intestine. Immunohistochemical study revealed 5-HT positive cells in the bottom of crypts and CD3- and CD 8-positive lymphocytes in lymphoid nodules. These findings suggest that the tree shrew has a cecum with primitive characteristics.

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