Proliferative Enteropathy caused by Lawsonia intracellularis in Chickens.

Proliferative enteropathy (PE) is an infectious disease caused by Lawsonia intracellularis (Li), an obligate intracellular bacterium. PE is endemic in swine herds and has been reported in a variety of mammals including horses, hamsters, rabbits, rats, guinea pigs, ferrets, foxes, dogs, sheep, deer and non-human primates. Avian cases are reported only in ratite birds, including emus and ostriches. Some studies show an absence of Lawsonia spp. infection in chickens. In this study, we performed morphological and bacteriological examinations on the intestines of two broiler chickens that had been condemned at a poultry slaughter plant in Japan due to intestinal haemorrhage, which was a result of focal coccidial enteritis. Histopathology revealed proliferation of the villous epithelium in the small and/or large intestines, especially the caeca, regardless of coccidial lesions. Warthin-Starry silver staining and immunohistochemistry using anti-Li monoclonal antibody revealed numerous bacteria and/or antigens in the villous epithelium. Scanning electron microscopy and transmission electron microscopy revealed the presence of curved rods, morphologically compatible with Li, in the apical cytoplasm of the epithelium. Polymerase chain reaction products specific for Li were amplified from DNA samples extracted from formalin-fixed and paraffin wax-embedded tissue. These results suggest that Li can cause PE, characterized by proliferation of the villous epithelium, in chickens.

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

A testis-specific antigen of the C57BL/6 mouse.

Spleen cells from female mice of the C57BL/6 strain iso-immunized with an homogenate from 3-week-old mice testis were fused with P3U1 cells. After cloning, two hybridomas, producing IgM, were obtained. Tissue specificity of the monoclonal antibody (Moab) in ascitic fluid was investigated by indirect immunofluorescence. Moab 1A1 reacted specifically with the cytoplasm of spermatogonia, spermatocytes and spermatids, but not with spermatozoa. Testicular antigen recognized by Moab 1A1 (Moab1A1-TA) was prepared by tissue sonication and then subjected to gel filtration. Moab1A1-TA detected in the void volume by ELISA was analyzed by SDS-PAGE and Western blotting. Immuno-staining of membrane filters revealed a broad area within the 45-205 kD range. Moab1A1-TA was treated with proteolytic enzymes, but no changes were observed after Western blotting. Thus, Moab1A1-TA was further digested by peptidases and glycolytic enzymes, electrophoresed using cellulose acetate membranes and immuno-stained with Moab 1A1. Evidence obtained from these experiments strongly suggests that Moab1A1-TA consists of an acidic peptide and a carbohydrate molecule. The antigenicity would be included in the carbohydrate epitope. Moreover, partial digestion of Moab1A1-TA by keratanase indicates that the lacto-series structure is included in the antigenic carbohydrate moiety.

High intra-abdominal pressure increases plasma catecholamine concentrations during pneumoperitoneum for laparoscopic procedures.

BACKGROUND: Laparoscopic procedures are associated with several complications, such as hemodynamic, respiratory, and endocrine complications. In our previous clinical study, plasma epinephrine and norepinephrine concentrations remained unchanged after the insertion of a Veress needle, but increased significantly immediately after insufflation with carbon dioxide into the peritoneum. The mechanisms for this increase are unknown. OBJECTIVE: To investigate whether gas insufflation during pneumoperitoneum affects plasma catecholamine concentrations during laparoscopic procedures. DESIGN: Experimental study in pigs. MAIN OUTCOME MEASURES: The plasma concentrations of epinephrine and norepinephrine were measured in the pigs before and after pneumoperitoneum. The mean arterial pressure, heart rate, cardiac output, and arterial blood gas levels were measured, and the systemic vascular resistance was calculated. INTERVENTION: Air, nitrous oxide, or carbon dioxide were insufflated in turn into the peritoneal cavity of supine pigs. Thereafter, carbon dioxide was insufflated into the peritoneal cavity while the pig was in the left lateral decubitus position, and then in the right lateral decubitus position. Measures were performed before pneumoperitoneum and at the intra-abdominal pressures of 10 mm Hg and 20 mm Hg. One hour of resting time was allowed between each procedure. RESULTS: As compared with baseline values, the plasma concentrations of epinephrine and norepinephrine remained unchanged at 10 mm Hg but increased significantly at 20 mm Hg regardless of the gas used for the pneumoperitoneum (P<.05). The type of gas and differences in the position of the animals had no effect on the plasma epinephrine and norepinephrine concentrations. CONCLUSIONS: Excessive intra-abdominal pressure, but not the type of gas or body position, increases plasma catecholamine concentrations during the insufflation of gas into the abdominal cavity. Therefore, excessive insufflation of the pneumoperitoneum should be avoided.

Apoptosis in the lymphatic organs of piglets inoculated with classical swine fever virus.

The involvement of apoptosis in the lymphatic organs of piglets infected with classical swine fever (CSF) virus was investigated. Piglets were inoculated with CSF virus and 3, 5, 7 and 10 days post inoculation (DPI), the thymus, spleen and lymph node were examined. In the thymus cortex, macrophages phagocytizing the nuclear remnants or apoptotic bodies increased after 3 DPI. Thymus atrophy due to the loss of the cortex increased markedly during the observation period. Compact and shrunken nuclei indicating apoptosis were observed in the spleen and lymph node. DNA fragmentation was detected in the nuclei of lymphocytes in the thymus, spleen and lymph nodes, as well as at sites of focal necrosis. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method, ultrastructural characteristics of apoptosis, i.e. margination of condensed nuclear chromatin, were observed in the lymphatic organs from 2 DPI onward. These results suggest that apoptosis is involved in the pathology of CSF.

Expression of proinflammatory cytokine mRNA in the lymphatic organs of adult and neonatal pigs.

Inflammatory cytokine mRNA expression in the lymphatic organs of neonatal, 1-month-old and adult pigs was compared. The mRNA expression of interleukin (IL)-1beta, IL-6, IL-18 and tumor necrosis factor (TNF)-alpha in the spleen, thymus, tonsil and popliteal and mesenteric lymph nodes was investigated by semi-quantitative RT-PCR. Stronger IL-1beta mRNA expression was observed in the 1-day-old and 1-month-old piglets than in the adult pigs. In thymus, tonsil and mesenteric lymph node, IL-1beta mRNA expression in 1-day-old piglets was stronger than in 1-month-old pigs. The expression of IL-6 mRNA in the 1-day-old and 1-month-old tonsil tended to be stronger than in the adult pigs. IL-18 and TNF-alpha mRNA expression was constant in all the samples examined. The expression of IL-1beta and IL-6 mRNA may reflect an inflammatory reaction against the exo- and endogenous foreign bodies occurring in the lymphatic organs, especially in the tonsil, of neonatal piglets.

Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.

Kupffer cells are sessile tissue macrophages that have a role in liver defense against endogenous endotoxins. Because little information is available on the role of bovine Kupffer cells, we developed a primary culture method to investigate the function of bovine Kupffer cells. Kupffer cells were isolated from the caudate lobe of calf liver by perfusion with collagenase and pronase. Then, the cells were purified by gradient centrifugation followed by counterflow centrifugal elutriation. With the methods, a mean number of 1.5 x 10(6) Kupffer cells with a final viability of over 98% was obtained from 1 g of the liver. Over 95% of the isolated cells were positive for non-specific esterase activity and had surface molecule of CD68. The cultured Kupffer cells expressed mRNAs of tumor necrosis factor-alpha, interleukin (IL)-1 alpha, IL-1 beta and IL-6 by stimulation for 3 h with lipopolysaccharide. The primary culture of bovine Kupffer cells could be useful to investigate the systemic inflammatory response in bovine liver.

Association of interleukin-6 in the cerebrospinal fluid during crisis of calf with ammoniated feed syndrome.

Ammoniated feed syndrome (AFS) in cattle is a neurotoxic syndrome caused by feeding specific ammoniated forage. To clarify the pathophysiology of AFS, we examined the association of interleukin-6 (IL-6) in the brain. By feeding milk either from cows fed such ammoniated forage or milk added with 4-methyl-imidazole, newborn calves showed a neurotoxic crisis of hyperexcitability, ataxia, muscle tremor, circling, roaring, epileptoid seizure, sweating and marked fever response. Although these calves had no pathological lesions in the brain, we detected a rise in IL-6 in the cerebrospinal fluid (CSF). Tumor necrosis factor-alpha (TNF-alpha) was not detected in the CSF. In the sera, IL-6 and TNF-alpha hardly changed during the experiment. Administration of recombinant human IL-6 into the lateral ventricle resulted in fever. Thus, we believe IL-6 in the CSF is related to the fever response in newborn calves with AFS.

Transperitoneal laparoscopic adrenalectomy: experience in 100 patients.

Between July 1992 and October 1996, 100 transperitoneal laparoscopic adrenalectomies were performed on 99 patients at our hospital and affiliated hospitals. The clinical diagnoses were primary aldosteronism (41 patients), Cushing's syndrome (15), pre-Cushing's syndrome (6), pheochromocytoma (7; 8 adrenal glands), adrenal cancer (2), nonfunctioning adenoma (22), myelolipoma (3), and complicated adrenal cyst (3). Ninety-seven glands were removed laparoscopically. The mean operative time was 240 +/- 76 (SD) minutes and the mean blood loss 68 +/- 80 mL for the series. The mean blood was 77 +/- 113 mL when the three operations that were converted to open surgery are included. The mean times for the return to a normal diet and unassisted ambulation were 1.3 +/- 0.6 and 1.4 +/- 0.8 days, respectively. The mean duration of the use of analgesics was 1.5 +/- 1.3 days, including the day of surgery. In contrast, in the latest 10 open adrenalectomies done at Kyoto University Hospital, the mean operative time was 186 +/- 53 minutes and the mean blood loss 220 +/- 170 mL. The mean times for return to a normal diet and for unassisted ambulation and the mean duration of the use of analgesics were 1.9 +/- 0.3, 2.9 +/- 1.1, and 2.9 +/- 1.7 days, respectively. Thirty-six operations, excluding one converted to open surgery, performed at Kyoto University Hospital were selected to look at the learning curve for transperitoneal laparoscopic adrenalectomy and evaluated for operative time and blood loss. The mean operative time and mean blood loss in the first 10 procedures performed at Kyoto University Hospital were 256 +/- 63 minutes and 89 +/- 57 mL; however, these values were reduced to 177 +/- 39 minutes and 48 +/- 32 mL in the next 10 procedures at the same hospital. Laparoscopic adrenalectomy via the transperitoneal anterior approach can be equivalent to open adrenalectomy in efficiency with a shorter convalescence.

Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation.

The morphological and biochemical characterisation of adult chicken hepatocytes in a serum-free culture are described. When cultured in positively charged plastic dishes, chicken hepatocytes formed a monolayer cell sheet. The monolayer morphology of these chicken hepatocytes was quite distinct from the spheroid shape of rat hepatocytes cultured under similar conditions. Electron microscopy showed that the cytoplasmic organelles of chicken hepatocytes were well preserved in vitro. Two-dimensional gel electrophoresis showed that the chicken hepatocytes secreted liver-specific proteins. Several enzymes of glucose-6-phosphatase, cytochrome P-450 or glutathione S-transferase, involved in metabolic and biotransformation pathways in the liver, were retained in the chicken hepatocytes in a serum free condition. These findings suggest that the primary culture of adult chicken hepatocytes with a serum-free culture system could be useful to study the hepatic metabolic pathway in the chicken and its response to various chemicals.

Hepatic lesions in young rabbits experimentally infected with rabbit haemorrhagic disease virus.

Twenty young rabbits (eleven 2-week-old and nine 4-week-old) were experimentally infected with rabbit haemorrhagic disease virus (RHDV) to clarify susceptibility. They were killed chronologically up to 96 hours post-inoculation (PI) and examined for lesions. All inoculated rabbits were clinically normal, but grossly minute white or grey spots were detected throughout the liver. Histologically, the lesions consisted of aggregates of lymphocytes, macrophages and heterophils, with or without acidophilic bodies and necrotic hepatocytes. Immunohistochemically, RHDV antigens were found in the degenerated hepatocytes and in macrophages. The cellular aggregates were considered to be a reaction to necrotic hepatocytes infected with RHDV. It was concluded that some hepatocytes are susceptible to RHDV in young rabbits.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis.

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.

Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11.

Genetically modified corn has been approved as an animal feed in several countries, but information about the fate of genetically modified DNA and protein in vivo is insufficient. Genetically modified corn Bt11 is developed by inserting a recombinant DNA sequence encoding insecticidal Cry1Ab protein from Bacillus thuringiensis subsp. kurstaki. We examined the presence of corn intrinsic and recombinant cry1Ab gene by PCR, and the Cry1Ab protein by immunological tests in the gastrointestinal contents of five genetically modified corn Bt11-fed and five nongenetically modified corn-fed pigs. Fragments of corn zein (242 bp), invertase (226 bp) and of ribulose-1,5-bisphosphate carboxylase/ oxygenase genes (1,028 bp) were detected in the gastrointestinal contents of both Bt11 and nongenetically modified corn-fed pigs. Fragments of recombinant cry1Ab gene (110 bp and 437 bp) were detected in the gastrointestinal contents of the Bt11-fed pigs but not in the control pigs. Neither corn intrinsic nor cry1Ab gene fragments were detected in the peripheral blood by PCR. The gastrointestinal contents were positive for Cry1Ab protein by ELISA, immunochromatography, and immunoblot; however, these methods did not work for blood and precluded conclusions about any potential absorption of the protein. These results suggest that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.

Involvement of apoptosis in the endotoxemic lesions of the liver and kidneys of piglets.

The involvement of apoptosis was evaluated in lesions of endotoxemic piglets. A single injection with E. coli O111:B4 lipopolysaccharide (LPS) induced foci of coagulative necrosis in the liver and kidneys. No significant change was observed in these organs at 1.5 hr after LPS injection, but at 6 hr, epithelial cells with chromatin condensation or fragmentation and apoptotic bodies were visible. Foci of coagulative necrosis were formed within 24 hr after LPS inoculation. In and adjacent to the necrotic foci, dead hepatocytes with nuclear condensation or fragmentation were scattered. These dead cells were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods. Electronmicroscopy revealed apoptotic cells with condensed or fragmented homogeneous nuclear chromatin, and necrotic cells with irregularly destroyed nuclei and cytoplasmic membranes. Apoptotic cell death were also observed in parietal cells of the stomach and lymphocytes in the lymphatic system. DNA ladders with approximately 200-bp multimers were observed in hepatic, renal and thymic samples prepared after 6 and 24 hr of LPS injection by agarose gel electrophoresis. These results suggest that apoptosis is involved in the pathology of swine endotoxemia.

Interferon alpha therapy for chronic active hepatitis type C after renal transplantation and allograft rejection.

An acute type rejection episode occurred in one of two patients treated with Interferon alpha (IFN alpha) for type C hepatitis (CHC). Histopathological examination of the graft kidney revealed focal cellular infiltration and chronic transplant glomerulopathy which showed acute or chronic type rejection. In spite of bolus administration of methyl-prednisolone, the elevation of serum creatinine level continued. After administration of anti-human lymphocyte globulin (AHLG), renal function improved, but urinary protein was still positive. Another patient had no episode of rejection during or after IFN alpha therapy.

[A case of retroperitoneal schwannoma associated with primary hyperparathyroidism].

A rare case of benign retroperitoneal schwannoma in a 47-year-old female is reported. She was initially diagnosed to have an adrenal tumor at the Prefectural Cancer Center Hospital and was subsequently admitted to this hospital. When she was admitted, she was diagnosed as undergoing a hypercalcemic crisis (s-Ca 9.2mEq/l). Serum intact-PTH was elevated, but urine cyclic-AMP and % TRP were almost normal. Gd-DTPA enhanced T1 weighted MRI showed a hyperintensity area between the trachea and the thyroid. A parathyroid tumor and a retroperitoneal tumor were excised completely. The final pathological diagnoses were determined to be a parathyroidal adenoma and a retroperitoneal Schwannoma. After a 6-month follow-up period no recurrence and no electrolytic imbalance were detected and the patient presently displays no further symptoms.

[Study on platinum concentration in internal iliac venous blood after iliac artery cisplatin infusion for invasive bladder cancer].

We evaluated the usefulness of internal iliac artery infusion of cisplatin (CDDP) in the M-VAC therapy for bladder cancer. CDDP (average 72 mg) was administered into the internal iliac artery with an infusion time of 15 minutes in 8 cases of bladder cancers. After the end of CDDP intra-arterial injection, we measured the total and free platinum (Pt) levels in the internal iliac venous blood and systemic venous blood. Both total and free platinum levels in the internal iliac venous blood were about 4 times higher than those in the systemic vein. No significant difference was found in total or free platinum level in the systemic blood between the intra-arterial and intra-venous infusion methods. Intra-arterial CDDP therapy is effective in the treatment of the local tumor and systemic micrometastasis.

[Yolk sac tumor of the testis in children: report of two cases].

Two cases of yolk sac tumor of the testis are presented. The patients were 17 months and 24 months old. The children were inflicted with painless swelling of their left scrotal content. alpha-Fetoprotein levels were elevated at presentation but decreased within normal limits after orchiectomy. Chest X-rays and CT scans were negative. The cases were diagnosed as stage I. Fifty six cases of testicular yolk sac tumor in children have been reported in Japan since 1981. There were no recurrent stage I cases. One patient with stage II and 3 patients with stage III died despite chemotherapy, while three children with stage II or stage III disease survived more than 36 months after a positive response to chemotherapy. We conclude that prepubertal stage I yolk sac tumor is treated best initially by orchiectomy alone. Aggressive chemotherapy has a major role in salvage of stage II or stage III patients.

[A case of renal vein thrombosis].

A case of renal vein thrombosis in a seventy-five year old female was reported. She complained of severe left flank pain. The symptoms and signs resembled obstruction from a ureteral calculus. The kidney-ureter-bladder X-ray showed a calcification in the pelvic cavity. She was admitted under the initial diagnosis of left ureteral stone. The venous phase of renal arteriography revealed venous collaterals (ureteric vein and gonadal vein). Selective renal phlebography demonstrated a radiolucent area. Warfarin, 6 mg orally daily, has been administered for a year. It has effectively prevented subsequent emboli. This was a rare case of renal vein thrombosis in an old patient, because it was not associated with nephrotic syndrome or thromboembolic state and because it presented as sudden onset.

[Traumatic dislocation of the testis].

Dislocation of the testis is a rare injury, with only 73 cases having been reported in Japan. We herein add 6 cases. Lately, accidents involving teenage patients, primarily involving motorcycles, have been increasing. Closed reduction is recommended, but it was successful only 5 of 73 cases. The results of testicular biopsies suggest that early surgical management is required when closed reduction is unsuccessful.