Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Polymeric immunoglobulin receptor expression and local immunoglobulin A production in bovine sublingual, submandibular and parotid salivary glands.

The submandibular and parotid glands are the main sources of immunoglobulins A (IgAs) in human and rat saliva. These glands express the polymeric immunoglobulin receptor (pIgR), which transports IgAs into saliva. The main source of IgAs in saliva and pIgR expression in salivary glands has not been well documented in cattle. Expressions of pIgR were determined in the major bovine salivary glands (sublingual, submandibular, and parotid) by RT-PCR for mRNA and by Western blot analysis and immunohistochemistry (IHC) using an anti-human pIgR antibody for protein. The protein detected with the antibody was identified by nano-liquid chromatography-quadrupole time of flight mass spectrometry. Additionally, the distribution of Ig-producing plasma cells was analyzed by IHC. RT-PCR showed that pIgR was expressed in the sublingual and submandibular glands, but not in the parotid gland. Higher protein levels were observed in sublingual glands than in submandibular glands by Western blot. By IHC, pIgR was mainly located on the apical side of the cytoplasmic membrane in the sublingual gland, whereas it was observed only on the basal side in the submandibular gland. The highest density of plasma cells expressing IgAs was observed in the sublingual gland. These results suggest that the sublingual gland plays an important role in first-line defence of the oral cavity in cattle in contrast to humans and rats.

Short communication: effects of serum obtained from dairy cows with low or high body condition score on in vitro embryo development.

The objective of the study was to determine whether the serum obtained from animals differing in body condition score (BCS) affects in vitro embryo development. After in vitro fertilization, serum obtained from dairy cows of either low (L-BCS; 2.1 ± 0.14 on a scale of 1 to 5) or high BCS (H-BCS; 4.0 ± 0.0), or commercially available bovine serum (control) was added at 5% concentration to the in vitro culture medium. Use of serum obtained from H-BCS cows increased the cleavage rates compared with control serum at both 24 and 48 h after in vitro fertilization (78.3 vs. 71.9% and 79.9 vs. 75.1%, respectively), whereas use of serum obtained from L-BCS cows increased the blastocyst rate compared with control serum at 7d (23.8 vs. 19.1%), but this difference was not evident at 8 or 9 d after in vitro fertilization. As nonesterified fatty acid concentrations were highest in control serum, followed by serum from L-BCS and H-BCS cows (621, 559, and 272 µEq/L, respectively), a high concentration of nonesterified fatty acids might adversely affect the very early stages of embryo development, and its negative effects might be greater immediately after fertilization compared with developmental stages after morula formation. Our findings also indicate that factors promoting early stage embryo development do not necessarily promote blastocyst development. Serum obtained from animals under different physiological conditions may be used for in vitro embryo culture to study the effects of nutritional management of dairy cattle on embryo development.

Hemophagocytic histiocytic sarcoma in a Japanese black cow.

At necropsy, an 11-year-old Japanese Black cow with anemia, leukocytopenia, and progressive hind limb ataxia had marked diffuse splenomegaly and multiple masses in the thoracic vertebrae. Histologically, neoplastic erythrophagocytic histiocytes were in the splenic red pulp, vertebral masses, and blood vessels of the liver and lungs. The spinal cord was compressed by the vertebral masses. Clinicopathological, macroscopic, and histologic findings were consistent with hemophagocytic histiocytic sarcoma. Vertebral involvement with spinal cord compression and resultant hind limb ataxia is an unusual presentation for this tumor, which has been described mainly in dogs and cats.

Epithelioid cells in mediastinal lymph nodes of cattle without cancer.

Epithelioid cells are found in lymph nodes in cases of metastatic carcinoma; however, epithelioid cells with benign features have also been discovered incidentally in lymph nodes. Epithelioid cells were observed in mediastinal lymph nodes of cattle without cancer during routine diagnostic necropsy. To explain this finding, the authors evaluated the prevalence and histopathologic, immunohistochemical, and ultrastructural features retrospectively using mediastinal lymph nodes from 110 cattle and found that 66 of the lymph nodes contained epithelioid cells. In all 66 nodes, most of the epithelioid cells were individual or aggregated in nodal sinuses; in 21 nodes, some epithelioid cells formed tubular structures. The individual and aggregated epithelioid cells were mostly considered to be mesothelial in origin by immunohistochemical and ultrastructural examination; presumably, they entered the lymph nodes via lymphatic vessels, because they were found in afferent lymphatic vessels and nodal sinuses. Although the presence of epithelioid cells in lymph nodes prompts suspicion of metastatic carcinoma, the epithelioid cells in these bovine lymph nodes did not disrupt nodal architecture, lacked atypia or mitotic figures, and did not invade nodal parenchyma.

Feeding activated charcoal from bark containing wood vinegar liquid (nekka-rich) is effective as treatment for cryptosporidiosis in calves.

The effect of activated charcoal containing wood vinegar liquid (Nekka-Rich) on Cryptosporidium parvum was evaluated in vitro and in vivo. First, the adsorption of C. parvum by the activated charcoal component of Nekka-Rich was examined. When mixed, C. parvum oo-cysts were effectively adsorbed by activated charcoal. Next, the effect of the wood vinegar liquid component of Nekka-Rich was assessed. Wood vinegar liquid had an antiprotozoan activity against C. parvum oocysts. Finally, the anticryptosporidial effect of Nekka-Rich was evaluated in calves experimentally infected with C. parvum. Six neonatal calves at 7 d of age were orally infected with 1 x 10(5) oocysts of C. parvum. When diarrhea was observed (on d 3 after the experimental infection), 3 calves received a milk replacer containing 10 g of Nekka-Rich at 8-h intervals for 4 consecutive days, and 3 control calves were given a milk replacer without Nekka-Rich at 8-h intervals for 4 consecutive days. Calves fed milk with Nekka-Rich showed recovery from diarrhea 1 d after the start of treatment. Significantly less fecal excretion of C. parvum oocysts was observed 1 d after treatment in calves fed Nekka-Rich. On d 2 from the beginning of treatment with Nekka-Rich (on d 5 after the experimental infection), oocysts were not isolated from fecal samples. On the other hand, nontreated control calves developed severe diarrhea, and fecal excretion of oocysts was consistently observed. Calves in both groups were killed for collection of samples, which were subjected to scanning electron microscopy. Scanning electron microscopy revealed adherence of C. parvum oocysts to the intestinal epithelial surface of nontreated control calves, whereas a lack of C. parvum adhesion was observed in calves fed Nekka-Rich. These results suggest that Nekka-Rich would be useful for the treatment of cryptosporidiosis in neonatal calves.

Dose response to vaginal administration of 1,25-dihydroxyvitamin D3 to cows.

It was previously reported that intravaginal (IVAG) administration of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) might be protective against bovine hypocalcaemia. In the present study, various doses of exogenous 1,25(OH)(2)D(3) were administered IVAG to ovariectomised cows, and the subsequent changes in the biochemical parameters of the blood were measured to assess the characteristics of vaginal absorption. Five cows received 1,25(OH)(2)D(3) IVAG at a dose of 0.125, 0.25, 0.5, or 1.0microg/kg of body weight (BW) or intravenously at a dose of 1.0microg/kg BW. Dosing was at intervals of at least two weeks in a 5x5 Latin square design. Vaginally administered 1,25(OH)(2)D(3) was absorbed in a dose-dependent manner. There was no correlation between the IVAG dose of 1,25(OH)(2)D(3) and subsequent changes in plasma calcium concentrations. The bioavailability of 1,25(OH)(2)D(3) administered IVAG at 1.0microg/kg BW was approximately 93%.

A new method for detecting the abomasal position and characteristics of movement at the onset of the left displacement of the abomasum in cows.

A new method has been developed by us to observe the movements of the abomasum by using a magnet and digital magnetometer. Four cows with left displacement of the abomasum underwent conventional correction by rolling without tacking. A doughnut-type magnet was sutured to the pyloric region in a routine operation. The same was done in three control cows. The position of the pyloric region was observed with a digital magnetometer from outside the cow's body. The magnets in the pyloric region of the control cows were located at the right side of the abdominal cavity at 10-30 cm anterior to the udder base, and moved slightly in various directions within the span of a day. On the other hand, the magnets in the pyloric region of cows with abomasal displacement moved widely in the abdominal cavity from the normal right side to the abnormal left front side. A large movement of the magnet from the normal right side to the abnormal left side of the abdominal cavity was observed within 12 h of the onset of abomasal displacement.

Technical note: Vaginal absorption of 1,25(OH)2D3 in cattle.

Although exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administered via intravenous, intramuscular, and oral routes has been tested for efficacy in preventing parturient hypocalcemia in dairy cows, there are few reports concerning intravaginal administration. For this study, 1,25(OH)2D3 was administered via the bovine vaginal lumen, and subsequent changes in blood chemistry, including levels of 1,25(OH)2D3 and several minerals, were measured to confirm vaginal absorption. Each of 5 heifers received a single intravaginal dose of 1 microg of 1,25(OH)2D3/kg body weight; a single control heifer received the ethanol carrier alone. In heifers receiving 1,25(OH)2D3, the plasma 1,25(OH)2D3 levels increased markedly from baseline (88.3 +/- 20.3 pg/mL) within 2 h and reached a peak at 6 h after treatment (1967.4 +/- 1139.6 pg/mL). Plasma Ca levels increased from baseline (10.4 +/- 0.4 mg/dL) to a peak of 11.96 +/- 0.7 mg/dL at 24 h. The levels of inorganic phosphate in plasma increased over time from 7.3 +/- 0.5 to 8.1 +/- 0.8 mg/dL by 6 h and were maintained at a plateau level (9.1 +/- 0.7 to 8.6 +/- 0.6 mg/dL) from 24 to 96 h after treatment. Plasma magnesium decreased from a baseline level of 2.1 +/- 0.1 mg/dL to a plateau level of 1.8 +/- 0.1 mg/dL, which was sustained from 24 to 48 h after treatment. The present study provides evidence of the absorption of exogenous 1,25(OH)2D3 from the bovine vaginal wall, as shown by the marked elevation of plasma 1,25(OH)2D3 levels by 2 h after administration, and indicates the possible utility of intravaginal administration of 1,25(OH)2D3 for prophylaxis of hypocalcemia.

Classification of abomasal displacement in cows according to histopathology of the liver and clinical chemistry.

Histopathological features of livers and blood chemical values in cows with abomasal displacement were investigated. Liver biopsy samples were collected during redressment operations in 92 cows with abomasal displacement, and the samples were stained with haematoxylin and eosin or periodic acid Schiff (PAS). Blood was collected for chemical tests. Livers were histopathologically divided into the following four types: normal histology cases (21%), fatty degeneration cases (36%), cloudy swelling cases (19%) and fatty degeneration cases with cloudy swelling (24%). The number of PAS-positive samples was significantly higher in the normal histology group and significantly lower in the severe fatty degeneration group and severe cloudy swelling group. Cows with fatty degeneration had significantly higher levels of serum 3-hydroxybutyric acid, non-esterified fatty acid and aspartate aminotransferase than did those with cloudy swelling or normal histology. The results indicate that the morbid conditions of cows with abomasal displacement can be classified into four types.

Relationship between serum TNF activity and insulin resistance in dairy cows affected with naturally occurring fatty liver.

To clarity the relationship between tumor necrosis factor (TNF) and insulin resistance in dairy cows affected with fatty liver, naturally occurring cases were investigated. The affected cows were classified into following three groups according to histopathologic findings of the liver: mild fat droplet deposition (group 1; n=11), severe fat droplet deposition (group 2; n=10), and cloudy swelling (group 3; n=8). Serum TNF activities in Group 2 (8.67 +/- 2.16 U/ml) and Group 3 (11.65 +/- 1.92 U/ml) were significantly higher than that in Group 1 (3.57 +/- 0.81 U/ml) (p<0.05). The insulin-tolerance tests showed that the insulin-stimulated glucose disposal rates (GDR) in Group 2 (27.6 +/- 7.8%) and Group 3 (15.8 +/- 9.1%) were significantly lower than that in Group 1 (41.7 +/- 9.8%). There was a significant negative correlation between serum TNF activity and GDR in affected cows (r=-0.56, p<0.01). These results indicate that serum TNF activity is correlated with insulin resistance in cows with fatty liver.

Inflammatory cytokines and antigen-responsive mononuclear cells in peripheral blood of cattle infected with Salmonella takoradi.

To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4+ T cells to CD8+ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4+ T cells in the infected cattle. In contrast, the numbers of gammadeltaT cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6. p<0.01). Serum TNF-alpha activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs. such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-y was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.

Acute phase response in naturally occurring coliform mastitis.

Changes in the activities of serum cytokines and in acute phase response were observed in dairy cows with naturally occurring coliform mastitis. Seven cows with severe mastitis showed systemic and mammary inflammatory response throughout the observation period, and 11 cows with mild mastitis recovered and were able to be milked within 3 days of onset of mastitis. Serum interleukin (IL)-I and tumor necrosis factor (TNF) activities were higher in the severe group than in the mild group at the first appearance of symptoms. Elevated IL-1 activity was evident in the severe group throughout the observation period. Serum alpha-1-acidglycoprotein (alpha1AG) concentration began to rise with the beginning of mastitis in the severe group, and peaked at 9 days. Serum haptoglobin (Hp) concentrations peaked at 3 days, and decreased gradually after 3 days in the severe group. These results showed that there are dynamic changes in serum IL-1 activity and in serum alpha1AG and Hp concentrations in cows with severe coliform mastitis.

Changes of immunological response after experimentally ozonated autohemoadministration in calves.

Changes in subsets of peripheral blood mononuclear cells and lymphocyte blastogenesis reaction were observed after ozonated autohemoadministration (OAHA) in calves. Ten healthy calves were used in this study. After OAHA, CD8+ cells, CD14+ cells and WCl+cells increased (p<0.05). The level of lymphocyte blastogenesis stimulated by PHA decreased after OAHA. A significant increase in serum IL-6 activity was observed in OAHA calves. These results suggested that OAHA induced immunological changes in calves.

Ankle arthrodesis in rheumatoid arthritis using an intramedullary nail with fins.

We describe an intramedullary nail with fin-like longitudinal ridges that we have developed for arthrodesis of the ankle in rheumatoid arthritis. Four fins with sharp tips were attached to the distal part of a cylindrical nail to stabilize the tibiotalar and subtalar joints. We used this nail in 15 feet of 15 patients with rheumatoid arthritis who were followed for an average of 34.9 months. Postsurgery, 13 patients were allowed to bear weight immediately, as tolerated, without immobilization. By 3 weeks, these patients were able to bear weight fully. Solid fusion of the ankle joint in an acceptable position and good clinical results were obtained in all patients. The only complications were two cases of delayed wound-healing.

Characteristics of blood chemical parameters in male and female quails.

The sexual difference in blood chemical tests and the correlation among these tests were investigated in 40 male and 40 female quails. Significant intersex differences were observed in serum total protein, total cholesterol, triglyceride, calcium, phosphorus, uric acid and glucose levels but not in alkaline phosphatase and aspartate aminotransferase levels. In the female quails, many of the blood chemicals that showed a significant sexual difference were strongly correlated with each other.

Comparative analysis of blood chemical values in primary ketosis and abomasal displacement in cows.

Blood chemical values, including ketone bodies, were measured in 25 cows with abomasal displacement (displacement group), 16 cows with primary ketosis (ketosis group), and nine normal controls to investigate the pathophysiology of abomasal displacement. Increases in aspartate aminotransferase, gamma-glutamyl transpeptidase, non-esterified fatty acid (NEFA), and ketone bodies (3-hydroxybutyric acid and acetoacetic acid) were observed in the displacement and ketosis groups. Total cholesterol increased significantly in the ketosis group but decreased in the displacement group. Glucose was significantly low and reversely correlated to ketone bodies in the ketosis group but was not low and was not correlated with ketone bodies in the displacement group. While NEFA was correlated to ketone bodies in the ketosis group, it was not in the displacement group. A correlation between the values of acetoacetic acid and 3-hydroxybutyric acid was seen in both the ketosis and displacement groups. The fact that blood chemical values in ketosis cows were clearly different from those in displacement cows suggest that the biochemical mechanism of ketogenesis is different between these two groups.

Detection of interleukin-1 beta in sera and colostrum of dairy cattle and in sera of neonates.

In order to obtain basic information about bovine interleukin-1 (IL-1 beta), levels of IL-1 beta in sera and milk of clinically normal mature Holstein cattle before and after parturition and in sera of newborn calves were examined by ELISA. The level of IL-1 beta was undetectable in sera of mature cattle around the time of artificial insemination, but the concentration gradually increased and reached a peak at parturition and then decreased again to an undetectable level. IL-1 beta in milk was detected on the day of parturition but not thereafter. IL-1 beta mRNA was detected by reverse transcription-polymerase chain reaction in the cells from milk collected during 20 days before and 2 to 3 days after parturition, but was not detected thereafter. Although IL-1 beta was not detected in all the sera of newborn calves, the concentration transiently increased with peak titers on day 3 and became undetectable by day 14 after birth. Newborns that showed serum IL-1 beta on day 3 had been fed on colostrum in which the IL-1 beta concentration was significantly higher than that in colostrum that had been fed to newborns having no detectable IL-1 beta on day 3. These results indicate that IL-1 beta is induced in association with pregnancy in healthy dairy cattle and that the cytokine might be transferred to neonates via colostrum.