D. Kwon

Chronologic Localization of Mycoplasma hyopneumoniae in Experimentally Infected Pigs

The chronologic localization of Mycoplasma hyopneumoniae was examined by in situ hybridization in experimentally infected pigs for a period of 35 days after intratracheal inoculation. M. hyopneumoniae DNA was detected in bronchial and bronchiolar epithelial cells from infected pigs at 7, 14, 21, and 28 days postinoculation (DPI) and in alveolar and interstitial macrophages and type I pneumocytes from infected pigs at 14, 21, 28, and 35 DPI. Strong hybridization signals for M. hyopneumoniae were detected mainly at the luminal surface of bronchial and bronchiolar lining epithelial cells. When a hybridization signal was detected at the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole also exhibited peribronchiolar lymphoid cuffing. These observations suggested that the presence of M. hyopneumoniae in different tissues could be due to a difference in the duration of the infection.

Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea.

A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STh), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease. Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins. Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively. Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively. Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.

Diarrhoea in nursing piglets associated with coccidiosis: prevalence, microscopic lesions and coexisting microorganisms.

A retrospective study was made of natural infections with Isospora suis in nursing piglets, recorded from April 1994 to May 1997, to determine the prevalence, microscopical lesions and other microorganisms associated with coccidiosis. One hundred and five (17.3 per cent) of the 605 nursing piglets submitted from 304 pig farms were diagnosed positive for coccidiosis. The affected piglets were from seven to 20 days old, with a mean age of 11.1 days. Coccidiosis occurred in each year but the incidence peaked in July (15 cases, 14.3 per cent), September (15 cases, 14.3 per cent), October (16 cases, 15.2 per cent) and November (18 cases, 17.1 per cent) and was lowest in May (no cases), August (two cases, 1.9 per cent) and June (four cases, 3.8 per cent). Histopathologically, villous atrophy resulting from the necrosis and sloughing of epithelial cells was a prominent feature of infection with I suis. In 49 5 per cent of the nursing piglets, other enteropathogens were identified, Escherichia coli (47.6 per cent) and transmissible gastroenteritis virus (3.8 per cent) being the most commonly diagnosed. Forty-five of 50 E coli isolates associated with coccidiosis tested negative by polymerase chain reaction for enterotoxigenic virulence factors, such as fimbriae and enterotoxins.

Capsular serotype, toxA gene, and antimicrobial susceptibility profiles of Pasteurella multocida isolated from pigs with pneumonia in Korea

a 145 kD toxin encoded by the chromosomal toxA gene are most often associated with atrophic rhinitis, whereas type A strains exempt of toxA are commonly associated with pneumonia, pleuritis or abscessation (Chanter and Rutter 1989). Although toxigenic strains have also been isolated from pneumonic lungs, the prevalence is low (Pijoan and others 1984, Iwamatsu and Sawada 1988). Antimicrobial therapy is used to reduce both the morbidity and mortality associated with swine respiratory disease (Wilson and others 1986). In Korea, antibiotics are commonly administered in the field to control pneumonic pasteurellosis. Early and adequate antibiotic therapy is critical in reducing morbidity and preventing the spread of pneumonic pasteurellosis. However, differences have been detected when comparing the minimal inhibitory concentration (MIC) data ofvarious antimicrobial agents from one country with those from another (Gutierrez Martin and Rodriguez Ferri 1993, Salmon and others 1995, Stephens and others 1995). Therefore, for the treatment to be effective, it is important to know the antimicrobial susceptibility ofP multocida that is isolated in each country. The aims of this study were first to determine the prevalence ofP multocida capsular serotype and toxA genotype, and secondly, to determine the antimicrobial susceptibility of P multocida as determined by the new standard procedures of the National Committee for Clinical Laboratory Standards (NCCLS) for antimicrobial susceptibility testing of animal pathogens (NCCLS 1997). Between 1995 and 1998, 230 isolates ofP multocida were isolated from 250 growing and finishing pigs showing severe respiratory symptoms from several different parts of Korea, which were submitted to the Department of Veterinary Pathology at the Seoul National University. All pigs were submitted live, and immediately upon receipt were euthanased for postmortem examination, whereupon specimens of lung tis-

In Situ Hybridization for the Detection and Localization of Swine Chlamydia trachomatis

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or ompl mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.

Detection and Localization of ApxI, -II, and -III Genes of Actinobacillus pleuropneumoniae in Natural Porcine Pleuropneumonia by In Situ Hybridization

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.

Pathogenesis and Chronologic Localization of the Human Influenza A (H1N1) Virus in Cotton Rats.

Objectives We aimed to evaluate the pathogenesis and chronologic localization of human influenza A (H1N1) virus in experimentally infected cotton rats. Methods The animals were intranasally inoculated with 107 plaque-forming units of A/Solomon Islands/3/2006 (H1N1) influenza virus and evaluated for pathogenicity for a period of 28 days. Virus replication kinetics and pathological properties were assessed chronologically. Acute antiviral responses were evaluated by mean of real-time polymerase chain reaction. Results Cotton rats infected with A/Solomon Islands/3/2006 virus lost weight until 6 days post-inoculation (DPI) and showed decreased activity until 3 DPI. At necropsy, focal areas of redness and consolidation of lungs were evident at 1, 2, and 3 DPI. Lung histopathology showed moderate to severe interstitial pneumonia, alveolitis and bronchiolitis. Influenza A specific viral protein was detected in bronchiolar epithelial cells, alveolar septa and pneumocytes. Influenza viruses were recovered from the lungs during the early period of infection and the titer peaked at 1 DPI. Viral proteins were detected from 4 hours to 6 hours DPI. These trends correlate with the up-regulation of mRNA expression of the IFN-α, Mx1, and Mx2 genes that play critical roles in the anti-influenza response at the early stage of infection. Conclusion Our results provide evidence that supports the use of cotton rats for the study of influenza virus pathogenesis and the immune response.