Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

Expression of P-glycoprotein and breast cancer resistance protein in three cases of canine lymphoma showing drug resistance

In canine lymphoma, drug resistance is the major factor hindering treatment. In this study, we performed immunohistochemical examination of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which are considered as transporters related to multidrug resistance in three recurrent canine lymphomas. All cases were negative for both transporters before anticancer drug administration, but became positive after this administration. The expression was confirmed in capillary endothelial cells, such as in brain capillaries acting as the blood-brain barrier (BBB). It is suggested that both transporters expressed on capillary endothelial cells in lymphoma tissue may inhibit the spread of anticancer drugs into tumor tissues from blood, the same as the BBB. Therefore, capillary endothelial cells could act as a blood-tumor barrier, which might be involved in drug resistance in canine lymphoma.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Effect of activation methods for bovine oocytes after intracytoplasmic injection

A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (i.c.i.). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after i.c.i., reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by i.c.i.     

Increased ectopic fat cells in the longitudinal muscularis layer of the oviduct isthmus in obese Japanese Black cows

In obese humans, mesenchymal stem cells differentiate to become ectopic fat cells in muscles. These ectopic fat cells inhibit the contraction of vascular smooth muscles. Stem cells have been recently identified in the human oviduct, a structure important in reproduction. We therefore investigated the number of Oil Red O (ORO)‐positive cells in the oviducts of control Japanese Black cows (n = 6; body condition score [BCS], 3.0 on a 5‐point scale) compared to those with diet‐induced obesity (n = 5; BCS, 4.0). We stained the ampulla and isthmus collected on the second day after ovulation with ORO and then counted the positive cells in each layer in 10 cross‐sections of the ampulla or isthmus. The obese group (23.4 ± 3.4 in the 10 sections) had larger numbers of ORO‐positive cells in the longitudinal muscularis of the isthmus (P < 0.05) than did the control group (15.0 ± 2.4). ORO‐positive cells were also observed in all other layers of the isthmus and ampulla; however, the number of cells in these layers did not differ significantly between obese cows and controls. Whether this observed increase in ORO‐positive cells in the oviducts of obese cows affects their reproduction warrants further study.     

How do visual and auditory cues influence calf stress reactions at weaning?

We investigated the effects of visual and auditory information related to cow and calf stress responses at weaning. Three weaning conditions were established with physical separation only (PS), physical and visual separation (VS), and physical, visual and auditory separation (VAS). Stress responses such as vocalization and self‐grooming, and changes of three maintenance behaviors (lying, walking and grazing) were recorded for experimental cows and calves in the respective conditions. Comparison of the data revealed that the cow responses differed slightly among groups. However, the peak frequency of vocalization was higher in the PS calves than in those of either VS or VAS. Self‐grooming frequency was significantly different in calves and was high in the order of VAS, VS and PS calves. Furthermore, the percentage of time spent lying down was greater, and that of walking was less, in VAS calves than in either VS or PS calves. These results demonstrate that different information at weaning might strongly affect the stress responses of calves but not those of cows. Relative impacts of different types sensory information on stressors must be considered to plan animal‐friendly weaning.     

Overcoming the self-incompatibility of Raphanus sativus by application of plant hormones,amino acids and vitamines,and by temperature treatment of pollen

Summary Overcoming self-incompatibility by application of three kinds of plant hormones, sucrose, 3 kinds of amino acids and 2 kinds of vitamines was tested in cvs. Honbashi-taibyo Minowase (H-Mino) and Minowase (Mino) of Raphanus sativus. Effects differed between the cultivars. In H-Mino, BA (100 mg/l) and glutamic acid, folic acid and nicotinic acid (500 mg/l) resulted in higher fruit set and higher number of seeds per pollinated flower. In Mino, BA and NAA (100 mg/l) and glutamic acid and glycine (500 mg/l) induced a high number of seeds per pollinated flower. These chemicals, however, induced parthenocarpic fruit set, especially GA3. From the observation of pollen on stigmas washed with glutamic acid, it appeared that the pollen-tube penetrated into a papilla cell after 1 hour and openings of papillae and detached pollen grains and tubes were found after 2 hours as the result of successful pollentube penetration of papillae. Pollen was heated at 50°C for 30, 45 or 60 minutes, at 60°C for 15, 30 or 45 minutes and at 70°C for 10, 20 or 30 minutes prior to self-pollination. In H-Mino, 60 and 70°C were effective, and expecially 60°C for 15 or 30 minutes resulted a higher percentage fruit set and more seeds per fruit. In Mino, although 50–70°C were effective, the mean number of seeds per pollinated flower was lower than in H-Mino.     

Earth tides can trigger shallow thrust fault earthquakes

We show a correlation between the occurrence of shallow thrust earthquakes and the occurrence of the strongest tides. The rate of earthquakes varies from the background rate by a factor of 3 with the tidal stress. The highest correlation is found when we assume a coefficient of friction of mu = 0.4 for the crust, although we see good correlation for mu between 0.2 and 0.6. Our results quantify the effect of applied stress on earthquake triggering, a key factor in understanding earthquake nucleation and cascades whereby one earthquake triggers others.