Epidemiology, pathology, and immunohistochemistry of layer hens naturally affected with H5N1 highly pathogenic avian influenza in Japan

Epidemiology, pathology, and immunohistochemistry were investigated in layer hens affected with H5N1 highly pathogenic avian influenza, which occurred for the first time in 79 years in Japan. The farm, which had a total of 34,640 chickens, experienced up to 43.3% mortality before the chickens were depopulated. Clinically, the affected chickens exhibited mortality without apparent clinical signs. Histologically, hepatocytic necrosis; necrosis of ellipsoids and follicles with fibrin in the spleen; necrosis with glial nodules in the brain stem, cerebrum, and cerebellum; necrosis of acinar cells in the pancreas; and necrosis of lymphoid tissues in intestinal lamina propria were seen. Occasionally, mild bronchiolitis, degeneration of smooth muscle fibers in the cecum, and mild tubulonephrosis were noted. Immunohistochemically, influenza virus antigens were detected often in the liver and spleen, heart, intestine, gizzard, proventriculus, and oviduct. In addition, antigens were seen also in the brain, kidney, pancreas, and ovary, but seldom in the lung and trachea. Virus antigen was mainly detected in the capillary endothelium and parenchymal cells. This suggests that virus excretion from the respiratory tract was not as prevalent as that from the digestive tract in the present cases.     

Differentiation and elimination of uterine natural killer cells in delayed implantation and parturition mice

To clarify the roles of uterine natural killer (uNK) cells in implantation and parturition, differentiation and elimination of uNK cells in the pregnant uterus was examined using artificial delayed implantation (DI) and delayed parturition (DP) mice. To prepare DI mice, pregnant mice were ovariectomized on the third day of pregnancy (D3) and treated with 2 mg progesterone daily. The same amount of progesterone was administered on D15 or D17 of normal pregnant mice at 24 h intervals until sampling to prepare DP mice. The uNK cells contained PAS-positive granules on D8 in DI mice. The uNK cells in DI mice were smaller in size, and differentiation of these cells was delayed compared to those of the control mice. From D19 to D21 in DP mice, the metrial gland was well developed and uNK cells were present. The number of uNK cell granules decreased on D21, and there were no uNK cells in the normal pregnant mice. This result suggests that differentiation of uNK cells is not directly related to implantation, but elimination of these cells is closely involved in parturition.     

Factor XI mutation in a Holstein cow with repeat breeding in Japan

Factor XI deficiency is an autosomal recessive coagulopathy in Holstein cattle. Affected cows have a tendency to show repeat breeding. Forty repeat breeding Holstein Friesian cows were selected and tested for the Factor XI mutation. Genomic DNA was isolated from the blood of the cows (n=40). Exon 12 of the Factor XI gene of the cows was amplified by PCR. One repeat breeding cow was heterozygous to the Factor XI mutation as indicated by the presence of two DNA fragments of 320 bp and 244 bp. The insertion of the 76 bp in the heterozygous cow was confirmed by DNA sequencing. The heterozygous cow was in her fourth lactation. She gave birth to male twins at the last calving. She was inseminated artificially four times after the last calving. Factor XI deficiency in cattle has been reported in different countries. However, no case was reported in Japan. This might be the first to report Factor XI mutation in Holstein cattle in Japan.     

Histological study of granulated metrial gland (GMG) cells in beige (DA-bg/bg) rats

To clarify the roles of granulated metrial gland (GMG) cells for successful pregnancy in rats, GMG cells in beige rats (genotype: DA-bg/bg), whose NK cells show lysosomal dysfunction because of abnormalities in cytoplasmic granules, were examined in mid- and late-pregnancy by light and electron microscopies. The GMG cells of beige rats were significantly less in number than those of the two controls (genotypes: DA-bg/+ and DA-+/+) in mid- and late-pregnancy, and this accompanied a low reproductive performance in the beige rats. The size of intracellular granules in the GMG cells of the beige rats was larger than for the two controls on each corresponding day of pregnancy. These results suggest that the activity of rat GMG cells and peripheral NK cells might be influenced by the beige gene, which is involved in reproductive performance.     

Picrotoxinin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides

The nature of the picrotoxinin receptor was studied using the central nervous system (CNS) of the American cockroach. It first was confirmed by using an electrophysiological technique that the abdominal nerve cord of the American cockroach was sensitive to picrotoxinin. By using a $\text{[}{}^3\text{H]α-dihydropicrotoxinin}$ binding test it was determined that the picrotoxinin receptor in CNS of this insect had a higher affinity toward picrotoxinin and heptachlor epoxide than the corresponding receptor in the rat brain. Also, the cockroach brain preparation had a higher percentage of specific binding in the total binding, making this material suitable for receptor studies. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 to 1.2 M sucrose at 100,000g contained the highest level of specific binding site. The receptor showed a sensitivity to all insecticidal cyclodienes tested, namely photodieldrin, oxychlordane, endrin, heptachlor epoxide, γ-chlordane, dieldrin, aldrin, heptachlor, and isodrin (expressed in the order of potency). Among four BHC isomers, the γ-isomer showed the highest potency to bind with this receptor.     

Improved survival of vitrified in vivo-derived porcine embryos

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.     

No straight path: roaming in both ground- and excited-state photolytic channels of NO3 → NO + O2

Roaming mechanisms have recently been observed in several chemical reactions alongside trajectories that pass through a traditional transition state. Here, we demonstrate that the visible light-induced reaction NO(3) → NO + O(2) proceeds exclusively by roaming. High-level ab initio calculations predict specific NO Λ doublet propensities (orientations of the unpaired electron with respect to the molecular rotation plane) for this mechanism, which we discern experimentally by ion imaging. The data provide direct evidence for roaming pathways in two different electronic states, corresponding to both previously documented photolysis channels that produce NO + O(2). More broadly, the results raise intriguing questions about the overall prevalence of this unusual reaction mechanism.     

Computed tomography and radiographic lymphography of the thoracic duct by subcutaneous or submucosal injection

A simple method of lymphography of the thoracic duct was investigated. Using three female beagles, contrast media were administered rectally, vaginally and into the perianal tissue. The administration sites were gently massaged, and imaging was carried out at constant intervals using computed tomography and radiograph. Moreover, Indian ink was administered into the rectum mucous membrane in dogs for proof of this method of lymphography, and the lymph drainage routes were observed. The investigation showed that clear computed tomography and radiographic contrast images of the thoracic duct were obtained by subcutaneous and submucosa injection of angiography contrast medium and 3D processing of these images revealed the three-dimensional positions and course of the thoracic duct and cisterna chyli.     

Adenosine and ATP affect LPS-induced cytokine production in canine macrophage cell line DH82 cells

Macrophages are essential for controlling the majority of infections, and are mediators of natural immunity. During infection, lipopolysaccharide (LPS) stimulates macrophages to produce pro-inflammatory cytokines. Adenosine and ATP released into the extracellular space by immunological stimuli have been shown to regulate various immune functions. More recently, it has been shown adenosine and ATP have a critical role on the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction between LPS, which generates the inflammation, and adenosine and ATP, which terminate the inflammation. We evaluate effects of adenosine and ATP on the production of cytokines related to inflammation in canine macrophage cell line DH82 cells. Adenosine and ATP respectively increased the production of IL-10 without affecting the production of IL-6, TNF-α and IL-12 in DH82 cells. In addition, adenosine and ATP prevented the production of LPS-induced IL-6, TNF-α and IL-12 in DH82 cells. In contrast, adenosine and ATP potentiated LPS-induced IL-10 production in DH82 cells. Moreover, adenosine, but not ATP inhibited LPS-induced expression of TLR4 in DH82 cells. These results suggest that conditions related to increased adenosine and/or ATP may play an important role in the inflammatory reactions.     

Estimation of glomerular filtration rate in calves using the contrast medium iodixanol

To develop a simple procedure for estimating glomerular filtration rate (GFR) in calves, a three-sample method using iodixanol was first compared to that using the standard agent inulin. Iodixanol and inulin were co-administered intravenously to calves at 40 mg I/kg and 40 mg/kg, respectively, and blood was collected 30, 60, 120, and 180 min later. Serum iodixanol and inulin concentrations were separately determined by high performance liquid chromatography and colorimetry. Serum urea nitrogen (UN) and creatinine concentrations were also measured. GFR estimated by iodixanol was consistent with that using inulin in clinically healthy calves. Based on GFR estimations in healthy calves and those renal-loaded with iodixanol, it was found that the serum creatinine concentrations became elevated when GFR decreased to 60% of the reference value. In contrast, serum UN concentrations fluctuated widely, presumably due to extra-renal factors. When GFR was estimated using the three-sample method and compared with the single-blood-sample method, 62/69 (90%) of samples tested were within the agreement plots. The results demonstrated that the single-blood-sample method using iodixanol may be useful in monitoring GFR in calves.