Identification of an antilymphocyte transformation substance from Pasteurella multocida.

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.

Patterns of Rectal Temperature and Shipping Fever Incidence in Horses Transported Over Long-Distances.

Shipping Fever is a transport associated syndrome seen in equids and bovines transported during long distances. The microbial profile and clinical signs vary between species, and in horses it is characterized by pharyngeal commensal bacteria and aerosolized particulate matter invading the lower airway due to compromised mucocillary clearance mechanisms during transports. This leads to pyrexia, pulmonary parenchymal disease, inappetence, and in severe cases pleuropneumonia. It has been shown that the incidence of transport-related pyrexia in horses increases with travel time and distance, however, this incidence rate has been expressed as the cumulative number of horses showing pyrexia with the length of travel time during road transport (cumulative percentage), which does not accurately reflect the actual temperature fluctuations and their patterns in relation to shipping fever. This study aims to demonstrate the individual fluctuations of body temperature variations during transport, particularly febrile changes. 53 Anglo-Arab and Thoroughbred horses aged 23-30 months were transported by road over different distances and durations (36-61 h; 1,492-2,921 km) in 3 investigations carried out in the spring and mid-summer in the northern hemisphere. The results showed that the incidence of fever (characterized by rectal temperature >38.6°C) was highest from 20 to 49 h after the start of transport. Clinical signs of shipping fever was observed in 25 of the 53 horses (47.2%), of which 10 horses (18.9%) exhibited fever at the end of transportation and 15 horses (28.3%) did not. This showed that horses that develop shipping fever do not necessarily present with fever at the end of transportation. Necropsy of 20 horses performed immediately after transportation suggested that transport induced pneumonia, contributed to the onset of pyrexia. This finding supports the suggestion that measuring body temperature upon arrival to determine the presence or absence of shipping fever could result in missed diagnoses for some horses with subclinical pneumonia, and that taking multiple temperature measurements at intervals from 20 h of transportation is a simple method for not missing horses with subclinical pneumonia.

Epidemiology of racing injuries in Thoroughbred racehorses with special reference to bone fractures: Japanese experience from the 1980s to 2000s.

This report describes the descriptive epidemiology of racing fractures that occurred from the 1980s to 2000s on racetracks of the Japan Racing Association (JRA). The incidence of racehorse fractures during flat racing was approximately 1-2%. Fractures occurring during a race are more likely to occur in a forelimb. Fractures mostly occur at the third and fourth corners of oval tracks and on the home stretch. They also occur more frequently at the time of changing the leading limb. Comparison of the incidence of racing fracture between before and after reconstruction of the geometrical configuration of a racetrack revealed that there was an outstanding reduction in the number of serious fractures in the year before and after reconstruction. It was postulated that the improvement in racing time, possibly influenced by reconstructing the geometrical configuration of the racetrack, was connected to the reduction in the number of fractures. Of non-biological race- and course-related factors, type of course (dirt or turf), track surface condition, differences between racecourses, and racing distance significantly influence racing time. By using an instrumented shoe, vertical ground reaction forces (VGRFs) on the forelimb during galloping and the relationships between a rough dirt and woodchip track surface and a smooth dirt and woodchip surface were measured. Relating the incidence of racing fractures with track conditions in general showed that track surface has significant effects on the incidence of fracture, with the incidence of fractures increasing as track conditions on dirt worsen and a tendency for the incidence of fractures to decrease as track conditions on turf worsen. It seems probable that track condition in general may affect the incidence of fracture. The incidence of fracture in horses during both racing and training decreased as the years progressed.

Histopathological characteristics of endometrosis in thoroughbred mares in Japan: results from 50 necropsy cases.

Uteri from 50 necropsied nonpregnant Japanese Thoroughbred brood mares (1-30 years of age) were investigated to clarify the histopathological characteristics of endometrosis in Japanese Thoroughbred mares and the distribution pattern of endometrosis lesions in the uterus as a whole. Endometrosis was observed in all animals over 6 years of age and in all of the 21 mares aged over 12 years of age. The affected mares showed elastofibrosis of arteries, veins and lymphatic vessels in the uterine wall, atrophy of the uterine smooth muscle layers and hyperplasia of collagen fibers among the smooth muscle fascicles of the myometrium, in addition to pathomorphologic features of endometrosis such as stromal endometrial fibrosis accompanied by endometrial atrophy, periglandular fibrosis and reduction of uterine glands. The severity of the histopathological changes increased with advancing age. Lymphatic vessels with elastofibrosis showed marked lymph congestion, leading to lymphatic edema. With increasing age, the extent of the distribution of these lesions tended to expand from focal to diffuse involvement of the entire uterus. Based on these findings, we speculate that aging plays a role in the pathogenesis of endometrosis; circulatory disturbances due to intrauterine angiosis or angiopathy, particularly reduction of the arterial blood supply and disturbance of venous drainage, resulting in a reduction of lymphatic drainage (lymphatic edema), are closely related to the onset and progression of endometrial fibrosis and myometrial atrophy with fibroplasia may result in myometrial hypofunction during the peri-implantation or puerperal period.

Comparison of cytokine mRNA expression in peripheral CD4(+) , CD8(+) and γδ T cells between healthy Holstein and Japanese Black calves.

Japanese Black (JB) calves are more susceptible to infectious diseases compared to Holstein (Hol) calves. To clarify the immunological differences between JB and Hol calves, expression of cytokine messenger RNA (mRNA) was examined using peripheral CD4(+) , CD8(+) and γδ T cells. Healthy calves, 24 from each species, were examined. Blood samples were obtained from calves at 1 week, 1 month and 3 months old, eight calves for each age of each species. Peripheral blood mononuclear cells were stimulated with phytohemagglutin (PHA), and T cell subsets were isolated by positive selection using magnetic cell sorting (MACS). Levels of interlekin (IL)-2, IL-4, IL-10 and interferon (IFN)-γ mRNA in three T cell subsets were analyzed. WC1-N1(+) γδ T cell percentages were significantly lower in JB calves at 1 week and 1 month of age compared to Hol calves. In addition JB calves had significantly lower IL-2, IL-10 and IFN-γ mRNA in WC1-N1(+) γδ T cells at 1 and 3 months of age, whereas there were no significant differences in cytokine mRNA of CD4(+) and CD8(+) cells between the two groups. Decreased cytokine mRNA and cell number of peripheral γδ T cells may affect negatively on the immune system of JB calves.

Effect of progesterone on Th1/Th2/Th17 and regulatory T cell-related genes in peripheral blood mononuclear cells during pregnancy in cows.

T helper (Th) cells play a central role in immune responses and new Th1/Th2/Th17 and regulatory T (Treg)-cell paradigm in pregnancy has developed. Progesterone (P(4)) is essential for the maintenance of pregnancy; however the effect of P(4) on Th1/Th2/Th17 and Treg paradigm is unclear in cows. We evaluated the effect of P(4) on the expression of Th1/Th2/Th17 and Treg-related cytokines, transcription factors, and P(4) receptors in the peripheral blood mononuclear cells (PBMCs) from 8 pregnant (163.1 ± 16.9 days of gestation) and 8 non-pregnant luteal phase cows. PBMCs were stimulated with P(4) at 0, 0.1, 1 or 10 µg/ml, and the mRNA expression of Th1, Th2, Th17 and Treg-related cytokines (IFN-γ, IL-4, IL-17 and TGF-ß), transcription factors (T-bet, GATA-3, RORC and Foxp3) and P(4) receptors (PGR, PGRMC1 and PGRMC2) were analyzed by real time RT-PCR. In both pregnant and non-pregnant cows, P(4) significantly inhibited the expression of IFN-γ and IL-17 dose-dependently, whereas P(4) did not affect the expression of TGF-ß and Foxp3. In addition, P(4) significantly decreased the expression of T-bet and RORC, and enhanced the expression of IL-4 in the pregnant cows, but this reaction was not found in the non-pregnant cows. P(4) tended to increase PGRMC1 in the pregnant cows but not in the non-pregnant cows, indicating that PGRMC1 may be involved in the regulation of the effect of P(4) during bovine pregnancy. These results indicate that P(4) is an important regulator of Th1/Th2/Th17 and Treg immunity, and higher Th2 immunity is characteristic in the pregnant cows.

Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares.

Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares.

Equine Endometrial Gland Density and Endometrial Thickness Vary among Sampling Sites in Thoroughbred Mares.

The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium. The aims of this study were to assess and compare the endometrial gland density and thickness at four sampling sites in the uterus (the central segment of each uterine horn, the uterine horn-body junction, and the caudal portion of the uterine body). Endometrial samples from five nulliparous Thoroughbred mares in diestrus were obtained at necropsy and used for subsequent histomorphometric analysis. The caudal uterine body had a significantly lower endometrial gland density and endometrial thickness than the other sites. This may result in nutrient deprivation and reduced survival of embryos or fetuses in this region of the uterus. The endometrial gland density and endometrial thickness did not significantly differ between the other regions sampled, indicating that they are similarly suitable for embryonic implantation and fetal development. Our results suggest that the endometrial structure of the caudal uterine body of the mare is not representative of the endometrial morphology at other sites. Thus, the caudal uterine body is not a suitable site for routine endometrial biopsy.

Comparison of the immunosuppressive effects of dexamethasone, flunixin meglumine and meloxicam on the in vitro response of calf peripheral blood mononuclear cells.

This study compared the immunosuppressive effects of dexamethasone (DEX), flunixin meglumine (FLU) and meloxicam (MEL) on the peripheral blood mononuclear cells (PBMCs) of seven healthy Holstein calves in vitro. DEX significantly inhibited lymphocyte proliferation and expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 messenger RNA (mRNA) in comparison with FLU and MEL. FLU and MEL dose-dependently inhibited lymphocyte proliferation, but did not significantly reduce mRNA expression. Our in vitro study indicates that steroidal anti-inflammatory drugs (SAIDs) as well as nonsteroidal anti-inflammatory drugs (NSAIDs) have immunosuppressive effects on calf PBMCs. These findings are important for assessing the indications and complications of NSAIDs in calves.

Successful embolization using interlocking detachable coils for a congenital extrahepatic portosystemic venous shunt in a child.

The authors report the case of a 6-year-old boy with a congenital extrahepatic portosystemic venous shunt. He had hyperammonemia. The shunt was 18 mm in diameter and located between the inferior mesenteric vein and the left internal iliac vein. The flow in the shunt was very rapid. After decreasing blood flow by inflating a balloon catheter inserted into the left internal iliac vein from the femoral vein, a microcatheter was coaxially advanced to the shunt to embolize the shunt. Embolization was successfully performed with interlocking detachable coils and microcoils without any complication. This patient's hyperammonemia resolved soon after the procedure.