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.

Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves.

A total of 378 isolates of Pasteurella multocida from clinically healthy and diseased calves were characterised for their susceptibility to 9 antimicrobial agents and screened by PCR for the presence of antimicrobial resistance genes and 22 genes virulence-associated, including capsule biosynthesis genes. Of the 378 isolates, 102 (27.0%) were resistant to at least one of the 9 tested antimicrobial agents. Resistance to oxytetracycline (21.7%) was the most frequently observed phenotype among the isolates. The tet(H) gene were the primary determinant detected. The resistance rates for thiamphenicol, ampicillin, kanamycin and florfenicol were 13.2%, 5.8%, 9.0% and 0.5%, respectively. Cefazolin, ceftiofur, cefquinome and enrofloxacin were effective antimicrobial agents, with no resistant isolates emerging over the course of the investigation. Most isolates were identified as capsular type A, only 6.3% belonged to capsular type D and no other capsular type was identified. Four of the virulence-associated genes (pfhA, tadD, tbpA and HAS) exhibited associations to the capsular type, and three (pfhA, tbpA and hgbB) were associated with the disease status of the animals. These virulence genes have been considered as epidemiological markers and are hypothesised to have a strong positive association with the outcome of disease in cattle.

Relationship between serotype and the antimicrobial susceptibility of Mannheimia haemolytica isolates collected between 1991 and 2010.

The antimicrobial susceptibilities and serotype distribution of 310 Mannheimia haemolytica isolates obtained from cattle with bovine respiratory disease during 2002-2010 were investigated. Of the 310 isolates, 198 (63.9%) were resistant to at least one of the 16 tested antimicrobial agents. The resistance rates for ampicillin, amoxicillin, dihydrostreptomycin, kanamycin, oxytetracycline, doxycycline, chloramphenicol, thiamphenicol, nalidixic acid, enrofloxacin, and danofloxacin were 20.3%, 14.5%, 43.5%, 23.5%, 24.8%, 21.9%, 23.2%, 23.9%, 47.1%, 18.7%, and 18.7%, respectively. Almost 90% of the isolates belonged to three serotypes (serotypes A1, A2, and A6), and the relative prevalence of serotype A6 increased significantly over the last decade. Compared with bacteria belonging to other serotypes, bacteria belonging to serotype A6 exhibited a significantly higher antimicrobial resistance rates (χ2 test, p<0.05). The results of this investigation provide useful information for understanding the serotype prevalence and antimicrobial resistance patterns of one of the major bacteriological agents implicated in pneumonic pasteurellosis.

A case of suspected lysosomal storage disease in a neonatal Japanese black calf.

A 5-day-old Japanese black calf was necropsied and intracytoplasmic vacuolations were histologically observed in many tissues. In the central nervous system, intracytoplasmic inclusions and vacuoles were found in neuronal cells. Intracytoplasmic inclusions were more conspicuous in the nuclei containing large nerve cells, especially in the brain stem and spinal cord. These inclusions were stained weak positive to positive with alcian blue, Giemsa, Luxol fast blue and periodic acid-Schiff stains but not with oil red O. Ultrastructurally, neuronal inclusions were observed in lysosomes and consisted of an amorphous electron-dense substance and occasional membranous structures. These findings seem to differ from the cases of bovine lysosomal diseases that have been reported, and this case may be another type of lysosomal storage disease.

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.

Detection of Cry1Ab protein in gastrointestinal contents but not visceral organs of genetically modified Bt11-fed calves.

The fate of insecticidal Cry1Ab protein was examined in the gastrointestinal (GI) contents and visceral organs of calves fed insect-resistant genetically modified maize Bt11. Twelve cross-breed (Japanese black x Holstein) calves were fed either Bt11 or non-genetically modified isoline maize for 90 d. Peripheral blood, rumen juice and feces were collected fortnightly, and GI contents and visceral organs were collected at slaughter at the end of the experiment. Samples were checked for Cry1Ab protein by immunological methods, and visceral organs were examined pathologically. Trace amounts of Cry1Ab protein were detected in the GI contents but not in the liver, spleen, kidney, muscle or mesenteric lymph nodes. No lesions were observed pathologically. Cry1Ab protein in the feces was degraded quickly at atmospheric temperature. These results suggested that only a trace amount of Cry1Ab protein survived passage through the GI tract but was not transferred to liver, spleen, kidney, lymph nodes or muscles.

Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLink CBH351.

We tried to detect DNA fragments derived from maize in the intestinal contents of pigs fed genetically modified (GM) StarLink CBH351 maize (SL) or non-GM maize. Intestinal contents of 8 SL and 8 non-GM maize-fed pigs were collected at slaughter, and the genes of the recombinant cry9C and the maize intrinsic zein (Zel) were assayed by polymerase chain reaction (PCR) 3 times with a total of 4 primer pairs of different expected lengths. The cry9C gene (either 103 or 170 bp) was detected in the rectal contents (with a frequency of 25-37.5%) and in the cecal contents (25-50%) of the pigs fed SL. In a similar fashion, the zein (Zel) gene (either 242 or 329 bp) was detected in the rectal contents (with a frequency of 31.3%) and in the cecal contents (25-37.5%) of pigs fed on SL non-GM maize. These results suggested that ingested DNA was not totally degraded, but is present in a form detectable by PCR.

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.

[Comparison of management of advanced cancer in various organs].

The management of advanced cancer presents the greatest challenge to physicians involved in oncology. There will usually be a large burden of disease; cure is unlikely; and the needs of the patient in terms of pain control and palliation will also be important over and above the direct treatment of the disease. Different issues will arise depending on the site and pathological type of the cancer. Increasingly over the past few years, treatment protocols and guidelines have been developed for different cancers, but these can only be rough guides rather than definite treatment recommendations. Additionally in most cancers advanced disease offers the opportunity for evaluation of new treatments in Phase II studies and other trials. With the new generation of molecular targeted therapies, such as EGFR inhibitors, striking results are being seen in advanced disease that compare favourably with what has been seen previously. Other agents such as those which attack the tumour vasculature may also have promise in this setting. Palliation is also an important aspect of the management of advanced disease, and pain control in particular is an important component of patient management. In summary, the treatment of advanced disease provides a test bed for new agents, but this need to develop better cancer therapies must be balanced against patient needs for a pain-free and comfortable end to life.

[A case of systemic fat embolism in mixed connective tissue disease associated with interstitial pneumonia during steroid treatment].

A 67-year-old man with a four-year history of mixed connective tissue disease (MCTD) associated with interstitial pneumonia was admitted to our hospital with a complaint of dyspnea and moist cough. Because the interstitial pneumonia was exacerbated, he was given high-dose steroid treatment (pulse therapy and sequential oral treatment of PSL 50 mg/day). After treatment his general condition showed some improvement, but then he suddenly died of acute respiratory failure. Autopsy disclosed fat emboli in the lungs, kidneys, liver and myocardium. The fat embolism may have been a consequence of the steroid treatment. Fat embolism should be taken into account as one of the causes of the acute respiratory failure in collagen vascular disease patients receiving steroid treatment.

[Platinum compounds in cancer therapy--past, present, and future].

Platinum cytotoxics play an important role globally in the management of solid tumours. Cisplatin sets the standard for efficacy in both regions with careful administration to reduce nephrotoxicity. Carboplatin is associated with neurotoxicity, but has become the leading product in the US due largely to the easier to manage toxicity profile. Both agents have been widely used in both registered and non registered indications and are frequently combined with other cytotoxics. In Japan, cisplatin has been used successfully at low doses in combination with 5-FU based regimens and appears to achieve a synergistic effect, but controlled data are not yet available. More recently oxaliplatin (Europe) and nedaplatin (in Japan) have been introduced, but their clinical roles in therapy have yet to be established. One of the limiting features of the first generation of platinum compounds is that a significant proportion of tumours develop cross resistance to platins due to either changes in uptake or excretion, intracellular detoxification or accelerated DNA repair. The forum discussed the possibility for the development of better new platinum compounds, A new platin agent which had lower toxicity and higher efficacy across a wide range of cancers without the development of resistance would be a significant step forward. If the tolerability profile was suitable, an oral formulation may improve the quality of life for patients but this must not be at the expense of efficacy. Even after the introduction of new target based drugs, platinum cytotoxics are likely to be used to reduce the tumour mass and in some cases can be expected to potentiate the effects of the new agents. In preclinical studies, ZD0473 has been shown to by-pass some major mechanisms of resistance and has the potential to achieve these objectives and is now being evaluated in clinical studies in both Japan and the West.

[A case of interferon beta-induced pneumonia].

A 58-year-old man had been treated with one intravenous injection of 120 mg of nimustine hydrochloride (ACNU), ten thrice-weekly doses of 3,000,000 U of interferon beta, and brain irradiation for cerebral glioblastoma. One month later he had fever, appetite loss, a productive cough and dyspnea. Chest radiography and CT showed diffuse, nonsegmental ground glass opacity in both lung fields. Hypoxemia and lung shadows were exacerbated day by day. Bronchoalveolar lavage revealed an increases in the total cell count and the percentages of lymphocytes and neutrophils, and a decrease of the CD 4/8 ratio. Interferon beta therapy was stopped, and steroid pulse therapy and prednisolone 40 mg administration were initiated. The symptoms, hypoxemia and lung shadows quickly improved. Reported cases of interferon beta-induced pneumonia are rare.

[Development of molecular targeting drugs for the treatment of cancer-therapeutic potential and issues to be addressed in global development].

A survey of cancer treatment in a sample of hospitals > 100 beds conducted in 1998 compared with experience in the US showed that good progress has been achieved in Japan in the screening and early treatment of gastric cancer, and that the prognosis for breast cancer is better than in the West. Although in the past, the cytotoxic therapies available to physicians in Japan vs the West have been different, recent acceleration of regulatory review will result in a convergence of treatment paradigms and some improvement in acute response in many tumour types. However, world wide there is a need for new improved therapies in all cancers evaluated. Particular needs are in the management of NSCLC, advanced disease and cancers which form micrometastases. The eventual hope is that cancer can be turned from a lethal disease into a chronic disease where patients maintain a good QOL. Apart from anti hormonal therapies, the usual approach has been to kill the cancerous cells. However, the new approaches to intervening in the growth and migration of cancerous cells or the host tissue response by molecular targeting offer the promise of achieving a step change in therapy. Although EGF tyrosine Kinase inhibitors such as ZD 1839 have been shown to cause a conventional tumour response in NSCLC, many of these new approaches are unlikely to show a short term response even if they have the capacity to affect tumour development and increase disease free survival. Some compounds will require combination therapy with a conventional cytotoxic or radiotherapy to show their full benefit. For conventional cytotoxics, the usual approach to development has been to select the maximum tolerated dose and then evaluate the efficacy in advanced disease. However, for the new approaches which will not have such severe dose limiting toxicities, it will be necessary to select a surrogate marker of the intended biological effect to select the optimal biological dose (OBD) and dose regimen in phase I/II studies for further evaluation in phase II or III studies which are designed to show the expected patient benefit. The tumour target, the stage of the disease and the possible need for concomitant therapy will also have to be considered according to the mechanism of action of the product.

Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus.

Eight pigs were inoculated subcutaneously with a highly virulent hog cholera virus (HCV) strain ALD. The infected pigs developed severe illness and became moribund on postinoculation day (PID) 7 or PID 10. Histologic lesions were characterized by severe generalized vasculitis, necrosis of lymphocytes, and encephalitis. HCV antigen was detected in crypt tonsilar epithelial cells, macrophages, and reticular endothelial cells of lymphoid tissues. Antigen localization corresponded well with histologic lesions. Five pigs were inoculated with less virulent HCV Kanagawa/74 strain and were euthanatized on PID 30. All five infected pigs recovered from the illness but became stunted. They also had a slight follicular depletion of lymphocytes, histiocytic hyperplasia, and hematopoiesis in the spleen. Less virulent HCV antigen was observed in the tonsils, kidneys, pancreas, adrenal glands, and lungs. Although antigen localization was less associated with histologic lesions, immunoreactivity was stronger than that in the pigs infected with the ALD strain of HCV. An almost complete loss of B lymphocytes was recognized in pigs infected with the ALD strain and was correlated with follicular necrosis in lymphoid tissues. Loss of B lymphocytes was not prominent in the pigs infected with Kanagawa/74 strain. The number of CD4+ and CD8+ T lymphocytes was significantly higher than that in the noninfected control pigs.

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.

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.

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.

[Efficacy of single-dose therapy with levofloxacin for acute cystitis: comparison to three-day therapy].

To investigate the safety, efficacy and recurrence-inhibiting effect of a single dose of levofloxacin (LVFX) for the treatment of acute uncomplicated cystitis in women, 56 females with acute uncomplicated cystitis between 17 and 73 years old were studied, based on the urinary tract infection (UTI) drug efficacy evaluation. The patients were divided into two groups; in Group A a single 200 mg dose of LVFX was administered, while in Group B, 100 mg of LVFX was administered twice a day for three days. The patients were re-examined three days later. The presence or absence of recurrence was surveyed by a questionnaire 3 months after the treatment. The efficacy rate on the third day after the administration in Group A and Group B was 96.9% (32 cases) and 95.8% (24 cases), respectively, and the recurrence rate within three months after administration 17.4% (4 in 23 cases) and 5.6% (1 in 18 cases), respectively. As for adverse drug reaction, abdominal pain was seen in one case, without a clear cause-effect relationship. Although the number of cases studied was small, no significant difference was seen between the single dose group and 3-day dose group in the safety, efficacy and recurrence-inhibiting effect of the new quinolone antibacterial treatment for acute uncomplicated cystitis in women, confirming the usefulness of the single dose treatment.

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.