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.

[Pleomorphic carcinoma of the lung; report of a case].

A 83-year-old male was referred to our hospital for further examination of abnormal shadow on chest radiography. Chest computed tomography (CT) showed a tumor mass in his right lung. Bronchoscopy brushing cytology revealed non-small cell lung carcinoma and right middle lobectomy was performed. Histological findings showed large cell carcinoma comprised of spindle cell component, finally diagnosing as pleomorphic carcinoma of the lung. Although he was diagnosed as pT2N0M0 (stage IA) after the operation, massive liver metastasis was found 7 months later. We report this case with references to the literatures on pleomorphic carcinoma of the lung.

[Pleomorphic carcinoma of the lung with idiopathic interstitial pneumonia; report of a case].

A 69-year-old man was referred to our hospital for further examination of the abnormal shadow on chest radiography. Chest computed tomography (CT) showed a tumor mass, 4 cm in size, in his right lung (S6) and interstitial pneumonia in the surrounding lung parenchyma Bronchoscopic brushing cytology revealed squamous cell carcinoma cells. Right lower and middle lobectomy was performed due to the metastasis to interlober lymph node. Histological findings showed squamous cell carcinoma comprised of spindle cell component, and there were also fibroblastic foci and fibroid thickness in the interstitium. Therefore he was diagnosed as pleomorphic carcinoma and usual interstitial pneumonia (UIP). About 7 months after the operation, the patient died of mainly multiple bone metastases. Pleomorphic carcinoma with UIP is very rare, so we report this case with references to the literatures.

[Pleomorphic carcinoma of the lung; report of a case].

A 66-year-old female complained of cough, and was referred to our hospital. Chest radiography and computed tomography (CT) showed a tumor mass near the right hilum and atelectasis of the middle lobe. Bronchoscopy revealed a whitish polypoid tumor obstructing the middle lobe bronchus. Histology by punch biopsy suggested adenocarcinoma Right upper and middle lobectomy was performed, due to the direct invasion of the tumor from the middle lobe to the upper lobe. Histological findings showed adenocarcinoma comprised of spindle cell component, finally diagnosing as pleomorphic carcinoma of the lung. After the operation, systemic chemotherapy, including paclitaxel and carboplatin was performed. About 42 months after operation, the patient died of multiple brain metastases.

Bacteriological characteristics of Staphylococcus aureus isolates from humans and bulk milk.

The aim of this study was to clarify the epidemiological association and bacteriological characteristics of human and animal Staphylococcus aureus isolates. Pulsed-field gel electrophoresis showed that pulsotypes (PT) of isolates from bulk milk differed from PT from human isolates, suggesting that there is no epidemiological association between isolates from these 2 sources. The absence of a common PT could result from the lack of contact between the sources. Methicillin-resistant S. aureus from human secretions and S. aureus from bulk milk in Japan consisted of 1 and 2 dominant clusters, respectively, whereas methicillin-susceptible S. aureus from humans consisted of assorted clusters. Isolates belonging to the dominant clusters showed the coagulase serotype, the capsule serotype, detection of exotoxin genes, and antimicrobial susceptibility. Isolates from bulk milk did not show the penicillin-binding protein 2a gene, and 252 of 275 isolates belonging to the 2 dominant clusters of bulk milk were susceptible to ampicillin, cefazolin, erythromycin, chloramphenicol, oxacillin, and vancomycin. Moreover, the LukM/LukF'-PV leukotoxin gene was detected in 233 of 275 isolates belonging to the dominant clusters in bulk milk isolates. These results support the hypothesis that a number of factors play a role in the adaptation of S. aureus isolates to specific hosts.

Molecular typing of Mannheimia (Pasteurella) haemolytica serotype A1 isolates from cattle in Japan.

Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods were applied for molecular typing of 130 Mannheimia (Pasteurella) haemolytica serotype A1 isolates obtained from 13 prefectures in Japan. These isolates were divided into 15 ApaI PFGE profiles that formed six distinct clusters (clusters A-F). Fifty-three (40.7%) isolates were classified in cluster B, and 20.0, 13.8, 12.3, 6.9 and 6.1% of isolates were in clusters E, A, F, D and C, respectively. The isolates of cluster B were differentiated into seven subtypes (B1-B7) and subtype B5 contained 63% (34/53) of isolates. RAPD revealed four banding patterns (types I-IV), and among 130 isolates 60.7% (79/130) of isolates were RAPD type I. All of the RAPD type I isolates were grouped into clusters A-C by PFGE. There was no relationship between molecular typing and geographic origin of these isolates. These results indicate that isolates of M. haemolytica A1 strain with various molecular profiles have already spread in Japan and may have caused sporadic infections.

Effects of dexamethasone on the pathogenesis of porcine circovirus type 2 infection in piglets.

To investigate the effect of immunosuppression on porcine circovirus type 2 (PCV2) infection, hysterectomy-produced, colostrum-deprived piglets were inoculated with the virus by the intranasal or intraperitoneal route, with or without dexamethasone (DEX) treatment. Eleven piglets aged 8 days were divided into four groups, namely group A (four animals given PCV2), B (three given PCV2 with DEX), C (two given sterile medium with DEX) and D (two given sterile medium). No significant clinical signs were observed. Histopathological and immunohistochemical examination revealed granulomatous inflammation and PCV2 antigen in the lymphoid tissues of group B piglets, but not in the other three groups. Flow cytometric analysis of peripheral blood lymphocytes showed a reduced number of CD4+ T cells in DEX-treated piglets (groups A and C). No differences between groups were observed in respect of the number of B cells, serum IgG concentration, or PCV2 antibody titre. These results indicate that DEX influenced the pathogenic effects of PCV2 infection in lymphoid organs, and that suppression of cell-mediated immunity may play a role in the aetiology of postweaning multisystemic wasting syndrome.

Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination.

Immunohistochemical characterization of calf pneumonia produced by the combined endobronchial administration of bovine herpesvirus 1 and Pasteurella haemolytica.

Ten calves ("group 4") were inoculated endobronchially with Pasteurella haemolytica 4 days after inoculation with bovine herpesvirus 1 (BHV-1). Four calves (group 3) were similarly inoculated with P. haemolytica alone, and three (group 2) with BHV-1 alone. All group 4 animals showed severe respiratory signs and had bilateral lobar pneumonia; one died 6 days after inoculation with P. haemolytica. Two types of pneumonic lesion were observed. One was characterized by interlobular and interstitial lymphatic thrombosis, fibrinous pleuritis and coagulative necrosis, and the other by necrotizing bronchiolitis with intranuclear inclusion bodies. The former type of lesion was associated with the presence of P. haemolytica antigen and the latter with the presence of BHV-1 antigen. The weight of infection of BHV-1 and P. haemolytica in bronchoalveolar (BAL) fluid was clearly reflected in the immunohistochemical demonstration of the corresponding antigens in BAL fluid cells. Group 4 calves differed from the calves of groups 1-3 in showing 10-1530 times more endotoxin in BAL fluid. These findings suggested that BHV-1 infection partly destroyed the clearance mechanisms of the respiratory tract epithelium and exacerbated the subsequent P. haemolytica infection.

Rapid molecular typing of Listeria monocytogenes by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) is a highly discriminating tool for molecular typing, but the conventional PFGE protocol is time consuming. This paper describes a rapid method of PFGE for Listeria monocytogenes that yields results within 2 days.

Molecular typing of Escherichia coli O157:H7 (H-) isolates from cattle in Japan.

A total of 77 Escherichia coli O157:H7 (H-) isolates from cattle in Japan were investigated by molecular biological methods. Most of these isolates (43 isolates) possessed the stx-2 gene, but not stx1. Fifteen bacteriophage types and 50 pulsed-field gel electrophoresis (PFGE) profiles were observed. One isolate was indistinguishable from the human outbreak strain by these methods. This indicates that cattle must be considered as a possible source of human E. coli O157:H7 infection in Japan.

Ofloxacin otic solution in patients with otitis media: an analysis of drug concentrations.

OBJECTIVE: To measure the concentration of ofloxacin in otorrhea, serum, and middle ear mucosa after topical administration of 0.3% ofloxacin otic solution. DESIGN: Study of 0.3% ofloxacin otic solution administered in a single dose of 0.5 mL in adults or 0.25 mL in children with chronic suppurative otitis media and perforated tympanic membrane, with serial sampling of otorrhea and serum up to 8 hours after dosing and middle ear mucosa up to 2 hours after dosing. SETTING: Three hospitals in Kagoshima, Japan. PATIENTS: Thirty-eight patients (age range, 3-81 years) with chronic suppurative otitis media and perforated tympanic membrane; 20 patients had sampling of otorrhea and serum and 18 patients (who required middle ear surgery) had middle ear mucosa and serum sampling. RESULTS: High concentrations of ofloxacin were measured in otorrhea samples taken immediately after dosing, followed by a rapid, nonlogarithmic decline. Elimination of the drug through otorrhea was believed to be related to loss from the application site with ear drainage, rather than to biologic mechanisms. Maximum concentration of ofloxacin in otorrhea was seen at the initial sampling time, 30 minutes after dosing, with concentrations measured up to the last sampling at 8 hours. Very low concentrations of ofloxacin were found in serum after topical administration of the drug. Concentrations were not detected in serum samples of most of the patients. The highest concentration measured was 10 ng/mL. Drug concentrations were detected primarily in samples obtained up to 1 hour after the dose was administered. Mucosal drug concentrations were highly variable, ranging from nondetectable to 602 pg/g. For the 6 bacterial strains isolated from the middle ear, the highest minimum inhibitory concentration of ofloxacin was covered by otorrhea drug concentrations measured at up to 8 hours after dosing in some patients. No adverse events were observed. No clinically significant adverse changes in laboratory test results or audiometric results were observed. CONCLUSIONS: Drug concentrations were high in otorrhea, very low or not detected in serum, and highly variable in middle ear mucosa. Nonbiologic loss of the drug with the ear drainage through the external auditory canal and eustachian tube was probably related to the high concentration in otorrhea samples. Drug concentrations in middle ear mucosa suggest that the drug reaches the infection site.

Characterisation of Escherichia coli O157:H7 (H-) derived from cattle using random amplification of polymorphic DNA analysis.

One hundred and two isolates of Escherichia coli O157:H7 derived from cattle were characterised by random amplification of polymorphic DNA (RAPD). Four different RAPD profiles were observed. Based on the combined results of RAPD typing and toxin genotyping, the isolates could be divided into six distinct groups.

Antigenic and genetic analyses of the hemagglutinin of influenza viruses isolated from pigs in 1993.

Three strains of influenza A virus (H3N2) were isolated from pigs in Hokkaido, Japan in 1993. The hemagglutinin (HA) antigen of the three isolates was related to that of recent H3N2 viruses of human origin. The reactivity patterns of two of the isolates (A/sw/Obihiro/1/93 and A/sw/Obihiro/2/93) with monoclonal antibodies to the hemagglutinin of A/Bangkok/1/79 strain were similar to that of the human H3N2 strain isolated in Hokkaido in 1988, while that of the other one (A/sw/Obihiro/3/93) was similar to human H3N2 strains of 1993. In the phylogenetic analysis based on the nucleotide sequences of the HA1 regions, the HA genes of the two isolates were shown to be closely related to those of human H3N2 viruses isolated between 1986 and 1988. The remaining one isolate was shown to be closely related to those of current human H3N2 viruses. We have also found serological evidence that the A/sw/Obihiro/1/93 virus is circulating extensively in Obihiro swine. It is clear from these findings that pigs were infected with the recent H3N2 influenza virus during the human epidemic and that the virus has been maintained in pigs for at least five years.

Prevalence of antibodies to type A influenza viruses in swine sera 1990-1994.

A total of 3,120 swine sera collected for the years 1990-94 were tested for the presence of hemagglutination-inhibition (HI) antibodies against swine (H1N1) and human (H1N1 and H3N2) strains of influenza virus. No HI antibody against the swine strains was recognized during 18 months, though a slight prevalence (1.5-9.2%) of the antibodies was observed over the entire period. A wide variance in the incidence (0-26.3%) of antibodies against the human H3N2 strains was observed among the swine population.

Antigenic and genetic characteristics of H1N1 human influenza virus isolated from pigs in Japan.

Two strains of influenza A virus were isolated from pigs in northern Japan in 1992. Serological tests showed that the haemagglutinin (HA) and neuraminidase (NA) antigens were more closely related to those of recent human H1N1 viruses than to those of swine H1N1 viruses. The HA and NA genes of isolate A/sw/Obihiro/5/92 were shown to be closely related to those of current human H1N1 viruses. Evolutionary trees constructed from nucleotide sequences showed that the HA and NA genes of A/sw/Obihiro/5/92 were apparently on a branch cluster containing human strains isolated between 1990 and 1992.

Effects of cyclosporin A on susceptibility and interferon-generating capacity in mice infected with Toxoplasma gondii.

Cyclosporin A (Cs-A), a potent immunosuppressant, was administered to mice to evaluate the role of T lymphocytes for the development of a protective immunity to an obligate intracellular protozoan parasite, Toxoplasma gondii. Although daily administration of various amounts of Cs-A for 7 days enhanced the host susceptibility at all doses employed, a dose-dependent manner of Cs-A treatment was not observed as far as the dosage regimens applied here are concerned; mice died at the same rate (40%) among the groups receiving various amounts of Cs-A. Cs-A treatment had a differential effect on the course of disease depending on how it was given in relation to infection. All mice receiving 50 mg of Cs-A per kg per day for 10 days from the beginning of infection eventually died of toxoplasmosis. Cs-A did not suppress the production of intereferon(IFN)-alpha/beta that was induced shortly after the infection, whereas it reduced greatly the ability of Toxoplasma-infected mice to produce IFN-gamma induced by stimulation with Toxoplasma lysate antigen (TLA). Moreover, the decrease in IFN-gamma production correlated with an increase in the parasitic growth in the peritoneal cavities of Cs-A-treated mice. These results suggest that the immunosuppressive effect of Cs-A on the primary Toxoplasma infection in mice is expressed by inhibiting the development of effector T cells responsible for the production of IFN-gamma.

Treatment of head and neck tumors by contact Nd-YAG laser surgery.

The contact Nd-YAG laser system with ceramic rods which has been developed by us was applied to animal experiments and clinical practice. It was confirmed that: 1) this method can be performed at low power of 6 to 8 W in contact laser incision of soft tissue and at 3 to 4 W in localized laser hyperthermia; 2) it permits accurate and precise incision because mis-shots of laser irradiation can be eliminated in the target tissue; 3) it causes less bleeding with minimal damage to adjacent tissue; and 4) it has remarkably high controllability. The contact Nd-YAG laser systems are very useful as one of the new modalities for surgical procedures as well as localized laser hyperthermia in head and neck tumors.