Yoshinari Katayama

Expression of vascular permeability factor (VPFNEGF) messenger RNA by plasma cells: Possible involvement in the development of edema in chronic inflammation

Edema occurs in some types of chronic inflammation such as nasal polyps, uterine cervical polyps and gastric hyper‐plastic polyps. However, the factors or cellular components involved in the development of edema in chronic inflammation remain to be clarified. Recently, the gene encoding vascular permeability factor (VPF) or vascular endothelial growth factor (VEGF) and the genes encoding its receptors (kinase insert domain‐containing receptor (KDR) and fms‐like tyrosine kinase‐1 [fit‐1]) have been cloned. VPF/VEGF induces vascular hyperpermeability and vascular endothe lial proliferation through KDR or fit‐1 receptors. As there is a possibility that VPF/VEGF may play a role in the development of edema in chronic inflammation, we examined the messenger (m) RNA expression of VPF/VEGF and its recep tors in nasal polyp tissues, which is an example of chronic inflammation with remarkable edema. Using northern blotting, all nasal polyp tissues examined expressed mRNA of VPF/VEGF and KDR. In situ hybridization revealed that VPF/ VEGF mRNA‐expressing cells were scattered in the edematous stroma of nasal polyps. In the adjacent sections, these cells showed the morphological features of plasma cells and expressed mRNA of immunoglobulin light chains. Human B cell leukemia and plasmacytoma cell lines expressed VPF/VEGF mRNA but human mast‐cell leukemia and T cell leukemia cell lines did not. The alternatively spliced pattern of VPF/VEGF transcripts observed in nasal polyp tissues was consistent with that in plasmacytoma cell lines. Taken together, the VPF/VEGF mRNA‐expressing cells in nasal polyps appeared to be plasma cells, suggesting that plasma cells may play an important role in the development of edema in chronic inflammation through the production of VPF/VEGF.

Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens

ABSTRACT Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 104 CFU/ml) of S. zooepidemicus.

Pharmacokinetics and tissue distribution of minocycline hydrochloride in horses

OBJECTIVE To determine the pharmacokinetics and tissue distribution of minocycline in horses. ANIMALS 5 healthy Thoroughbred mares for the pharmacokinetic experiment and 6 healthy Thoroughbred mares for the tissue distribution experiment. PROCEDURES Each mare was given 2.2 mg of minocycline hydrochloride/kg, IV. Blood samples were collected once before minocycline administration (0 hours) and 10 times within 48 hours after administration in the pharmacokinetics study, and 24 tissue samples were obtained at 0.5 and 3 hours in the distribution study. RESULTS No adverse effects were observed in any of the mares after minocycline administration. The mean+/-SD elimination half-life was 7.70+/-1.91 hours. The total body clearance was 0.16+/-0.04 L/h/kg, and the volume of distribution at steady state was 1.53+/-0.09 L/kg. The percentage of plasma protein binding was 68.1+/-2.6%. Plasma concentration of free minocycline was 0.12 microg/mL at 12 hours. Minocycline was not detected in brain tissue, CSF or aqueous humor at 0.5 hours; however, it was found in all tissues, except in the aqueous humor, at 3 hours. CONCLUSIONS AND CLINICAL RELEVANCE Clearance of minocycline in healthy mares was greater than that reported for humans. For effective treatment of infections with common equine pathogens, it will be necessary to administer minocycline at a dosage of 2.2 mg/kg, IV, every 12 hours. This drug could be useful for infections in many tissues, including the CNS. The pharmacokinetic and tissue distribution data should aid in the appropriate use of minocycline in horses.

Postoperative Clostridium difficile infection with PCR ribotype 078 strain identified at necropsy in five Thoroughbred racehorses

Clostridium difficile is an important cause of acute enterocolitis in horses. We describe five cases of C difficile infection occurring postoperatively in Thoroughbred racehorses. Following diarrhoea or colic accompanied by a marked increase in packed cell volume (to ≥60 per cent) and leucopenia (≤4000 cells/μl) within two to four days after surgery in all five horses, four of them died or were euthanased because of colitis or severe diarrhoea. In these four horses, necrotising entero-typhlo-colitis was revealed by postmortem examination, and C difficile was recovered from the contents of the small and/or large intestine. The remaining horse was euthanased because of marked decline in general condition and the presence of a lung abscess, from which C difficile was isolated. The horse had had severe postoperative diarrhoea before the onset of respiratory disorder; laboratory tests for C difficile were not performed on the faeces. All C difficile isolates were toxin-A-positive, toxin-B-positive and actin-specific ADP-ribosyltransferase (CDT)-positive. The isolates were indistinguishable by pulsed field gel electrophoresis analysis, PCR ribotyping, and slpA sequence typing, and the slpA sequences and PCR ribotype patterns were identical to those of known PCR type 078. This case sequence might have been healthcare-associated infection, although there was about a four-month interval between each disease onset.

Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis

ABSTRACT Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis. IMPORTANCE Antibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Use of loop-mediated isothermal amplification to detect six groups of pathogens causing secondary lower respiratory bacterial infections in horses.

Microbial substitution occasionally occurs following the administration of antimicrobials to horses that have pneumonia or pleuropneumonia. Four specific loop‐mediated isothermal amplification (LAMP) assays were developed to detect some equine respiratory pathogens, namely strains of the Bacteroides–Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Staphylococcus aureus. These four LAMP assays and two previously published LAMP assays targeting Escherichia coli or Pseudomonas aeruginosa were used on clinical respiratory specimens and a high accordance found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials.

Abortion in a thoroughbred mare associated with an infection with avirulent Rhodococcus equi

An eight-year-old thoroughbred mare with no previous history of illness aborted a fetus at 196 days of gestation, and its internal tissues were examined immunohistologically and bacteriologically. The placenta was not examined, but specimens of the intrauterine fluids and the dams faeces were collected four days after the abortion and examined bacteriologically. No significant histological lesions were found in the fetus but the amnion and the umbilical cord were oedematous and had petechial haemorrhages. Rhodococcus equi was isolated in pure culture from the lung, heart and stomach contents of the fetus and from an intrauterine specimen and faeces of the dam. The anti-R equi antibody titre of the mare was high after the abortion. The diagnosis was confirmed in the lung of the fetus by immunohistochemical staining with R equi-specific antibodies. Isolates from the fetus and mare were identified as avirulent R equi by pcr and the mouse pathogenicity test. The avirulent isolates were characterised by pulsed-field gel electrophoresis, which yielded only one VspI profile in all the isolates from the fetus and its dam.

Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses

Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen–containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen–containing spirochetes were found in 7 of 12 horses (58.3%) and were more frequent in the cecum than in other parts of the bowel. It was not clear whether the infection was clinically related to diarrhea or dysentery, but histopathology revealed a close association between the bacterial infection and epithelial hyperplasia. Crypt epithelium consisted mainly of goblet cells and showed frequent mitosis throughout its length. Inflammatory cells and congestion were also present. There were numerous spirochetes in the crypts, and some invaded the cecal and colonic epithelia and underlying lamina propria. Ultrastructurally, the spirochetes were divided into 4 types. Three types were identified in degenerative epithelial cells or intracellularly. Brachyspira antigen–containing intestinal spirochetes invading the mucosa were capable of causing epithelial hyperplasia in the cecum and colon in the horses. The findings in this study will increase awareness of the importance of intestinal spirochetosis and may also be helpful for diagnosis and treatment of this condition.

Time-related Pathological Changes in Horses Experimentally Inoculated with Equine Influenza A Virus

To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.

Histopathological Characteristics of an Ossifying Fibroma Formed in the Maxilla of a Racehorse

A 1-year-old male thoroughbred racehorse experienced swelling of the left upper lip. The swelling was attributable to enlargement around the incisive bone of the interdental space posterior to the third incisor in the left maxilla. Even after two operations to reduce the bulk of the mass, it continued to increase in size. Dyspnea caused by stenosis of the nasal cavity forced us to perform euthanasia, and a pathological examination was conducted. Macroscopic examination of a section of the mass revealed the formation of multiple areas of solid fibrous tissue, and trabeculae within the incisive bone which had displaced the cortical bone. On histology, the mass was composed of trabecular bone-like structures due to the proliferation and aggregation of fibroblasts. Therefore, we diagnosed it as an ossifying fibroma. Equine ossifying fibroma is characterized by development in the mandible, but was formed in the maxilla in this case. Equine ossifying fibroma has not been reported previously in Japan. This is the first case of equine ossifying fibroma identified in Japan.