Time-dependent airway epithelial and inflammatory cell responses induced by influenza virus A/PR/8/34 in C57BL/6 mice

The present study examines the kinetics of airway epithelial remodeling and inflammation in the airways of C57BL/6J mice infected with influenza virus A/PR/8/34 (PR8). Mice were intranasally instilled with 50 plaque forming units (pfu) of virus or its respective vehicle, saline, and then were sacrificed at 3, 7, 10, 15, or 21 days postinfection (dpi). PR8 treatment resulted in airway epithelial cell regeneration as suggested by proliferating cell nuclear antigen (PCNA) positive staining at 7 and 10 dpi and mucous cell metaplasia (MCM) evident at 10, 15, and 21 dpi. PR8 treatment resulted in a classic pattern of inflammation observed in bronchoalveolar lavage fluid (BALF), in which neutrophils peaked at 3 and 7 dpi and monocytes, lymphocytes, and eosinophils peaked at 10 dpi before returning to background levels of detection. Chemokine (MCP-1) and cytokine (IL-6, TNF-alpha, IFN-gamma, IL-5, IL-4, and IL-9) levels peaked at 7 dpi in BALF. IL-13 levels were unaffected by PR8 treatment. Concurrent with inflammation, MUC5AC gene expression was markedly increased by PR8 treatment at 7 dpi. Collectively, the results of this study indicate that the onset of MCM in airway epithelium occurs during the remodeling process and persists after the inflammatory response has diminished.     

Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs

The aim of this study was to elucidate the pathogenesis of experimental infection with Korean type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the virus distribution, sites of viral replication, viraemia and gross and microscopical lesions in conventional pigs studied for 28 days after intranasal inoculation. Mean rectal temperature was significantly higher in infected pigs than in negative control pigs at 2 days post inoculation (dpi) (P=0.004), 3 dpi (P<0.001), 4 dpi (P=0.003) and 5 dpi (P=0.034). The log(10)TCID(50)/ml of type 1 PRRSV increased significantly at 0-1 dpi (P=0.024) and 5-7 dpi (P=0.029), but decreased at 10-14 dpi (P=0.026) and 14-21 dpi (P=0.012) in infected pigs. Infected pigs developed multifocal, tan-mottled areas of lung tissue with irregular and indistinct borders. Microscopical lesions, when present, were multifocal, mild to moderate, generally most extensive at 5-7 dpi (P=0.036), and were nearly resolved at 28 dpi. Type 1 PRRSV nucleic acid and antigen were detected exclusively within the cytoplasm of macrophages and type I and II pneumocytes. The score for PRRSV-positive cells increased at 3-7 dpi (P<0.05) and decreased at 10-14 dpi (P=0.034) in infected pigs. Thus, respiratory disease was reproduced in conventional pigs by infection with Korean type 1 PRRSV.     

In the absence of endogenous gamma interferon, mice acutely infected with Neospora caninum succumb to a lethal immune response characterized by inactivation of peritoneal macrophages.

Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-gamma)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-gamma-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-gamma-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-gamma lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-gamma-deficient mice were activated by exogenous IFN-gamma associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-gamma-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-gamma-deficient mice did not. However, in vivo treatment with exogenous IFN-gamma induced up-regulated MHC class II expression in IFN-gamma-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-gamma and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-gamma-deficient mice. The levels of IL-12 in IFN-gamma-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-gamma production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses against N. caninum.     

Interleukin-10, interleukin-12, and interferon-gamma levels in the respiratory tract following mycoplasma hyopneumoniae and PRRSV infection in pigs

The cytokine profile associated with either a T helper 1 (Th1) or Th2 response in a porcine respiratory disease model was assessed by measuring IL-12, IL-10 and IFN-gamma using RT-PCR and ELISA, respectively. IL-10, IL-12, and IFN-gamma levels in pulmonary alveolar macrophages and bronchial lavage fluid were increased in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, or both pathogens. At 10 days post-infection (DPI), both IL-10 and IL-12 mRNA levels were increased in both groups infected with PRRSV. The IL-12 levels were increased in pigs infected with both pathogens and IFN-gamma protein levels were increased in pigs infected with PRRSV alone and only numerically increased in the dual infection. At 28 DPI, IL-12 mRNA levels and IL-10 protein levels were increased in all infected groups. The mRNA level of IL-12 remained elevated in the group infected with both pathogens at 42 DPI. Production of IFN-gamma did not appear to be closely correlated with elimination of virus from the respiratory tract. However, when the virus existed in the lung, the local IFN-gamma production appeared to increase. Although IL-12 mRNA levels were significantly elevated in the pigs infected with both pathogens, the increased protein levels of IL-12 may compromise the immune system's ability to clear PRRSV from the lung. This could explain the prolonged presence of PRRSV, IFN-gamma production and the increased pneumonia observed in the lungs of dual-infected pigs. The increased levels of cytokines associated with both Th1 and Th2 responses in the respiratory tract of pigs infected with PRRSV and M. hyopneumoniae provides valuable information on the pathogenesis of these diseases.     

Cross-species infection of specific-pathogen-free pigs by a genotype 4 strain of human hepatitis E virus

Hepatitis E virus (HEV) is an important pathogen. The animal strain of HEV, swine HEV, is related to human HEV. The genotype 3 swine HEV can infect humans and genotype 3 human HEV can infect pigs. The genotype 4 swine and human HEV strains are genetically related, but it is unknown whether genotype 4 human HEV can infect pigs. A swine bioassay was utilized in this study to determine whether genotype 4 human HEV can infect pigs. Fifteen, 4-week-old, specific-pathogen-free pigs were divided into three groups of five each. Group 1 pigs were each inoculated intravenously with PBS buffer as negative controls, group 2 pigs similarly with genotype 3 human HEV (strain US-2), and group 3 pigs similarly with genotype 4 human HEV (strain TW6196E). Serum and fecal samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days postinoculation (dpi) and tested for evidence of HEV infection. All pigs were necropsied at 56 dpi. As expected, the negative control pigs remained negative. The positive control pigs inoculated with genotype 3 human HEV all became infected as evidenced by detection of HEV antibodies, viremia and fecal virus shedding. All five pigs in group 3 inoculated with genotype 4 human HEV also became infected: fecal virus shedding and viremia were detected variably from 7 to 56 dpi, and seroconversion occurred by 28 dpi. The data indicated that genotype 4 human HEV has an expanded host range, and the results have important implications for understanding the natural history and zoonosis of HEV.     

Development of in-situ hybridization for the detection of Mycoplasma haemosuis (Eperythrozoon suis) in formalin-fixed, paraffin wax-embedded tissues from experimentally infected splenectomized pigs

Mycoplasma haemosuis DNA was detected in experimentally infected splenectomized pigs by in-situ hybridization (ISH) with a nonradioactive digoxigenin-labelled DNA probe. An 839 base pair DNA probe targeting a 16S rRNA gene was generated by the polymerase chain reaction. Eight 6-week-old pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-infected pig blood and eight negative control pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-free blood. Two pigs from each group were killed for examination at 3, 7, 15 and 30 days post-inoculation (dpi). Red blood cells infected with M. haemosuis were first detected by light microscopy at 3 to 7 dpi. No M. haemosuis was observed in negative control pigs. Hybridization signals were evident in blood from the infected pigs at 3 dpi. The ISH method developed in this study was useful for the detection of M. haemosuis DNA in formalin-fixed, paraffin wax-embedded tissues and may be valuable for studying the pathogenesis of M. haemosuis infection.     

Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection

The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E(rns) and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E(rns)E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E(rns) fragment (E(rns)(aa 109-160)) or an E2 fragment (E2(aa 1-221)) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E(rns)E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi.     

Pathogenesis of swine influenza virus subtype H1N2 infection in pigs

The purpose of this study was to elucidate pathogenesis and viral distribution in pigs infected with swine influenza virus subtype H1N2, over a period of 10 days, by morphometric analysis and in-situ hybridization. Fifteen colostrum-deprived pigs aged 3 weeks were inoculated intranasally with virus. Pneumonia was severe at 1 day post-inoculation (dpi), moderate at 3 and 5 dpi, and mild at 7 and 10 dpi. The pulmonary lesion score was correlated with the score of cells positive by in-situ hybridization for swine influenza virus (r(s)= 0.9114, P< 0.05). The distribution of swine influenza virus varied according to the duration of infection. At 1 and 3 dpi, hybridization signals were detected mainly in the bronchial and bronchiolar epithelial cells, but they were detected mainly in the pneumocytes and macrophages (alveolar and interstitial) at 7 and 10 dpi. The results confirmed that swine influenza virus subtype H1N2, isolated in Korea, is a virulent pathogen causing severe pneumonia.     

Detection of classical swine fever virus in the ovaries of experimentally infected sows

Six sows were infected intranasally with a Korean isolate of classical swine fever virus (CSFV). The distribution of virus in ovarian tissues was then assessed for 21 days by in-situ hybridization and immunohistochemistry. CSFV was detected in the ovaries between 7 and 21 days post-inoculation (dpi) by both methods, but the labelling was particularly intense and widespread at 7 dpi. CSFV nucleic acid and antigen were located almost exclusively within the cytoplasm of cells shown by haematoxylin and eosin staining to be macrophages, which were numerous in atretic follicles. Small numbers of CSFV nucleic acid-positive cells with distinctly round morphology and oval nuclei, resembling monocytes, were also observed in the blood vessels of sows at 7 and 14 dpi. CSFV nucleic acid and antigen were not observed in primordial, primary or secondary follicles from infected sows at 7, 14 or 21 dpi. The results suggest that CSFV replicates in circulating peripheral monocytes and gains access to ovarian tissues from the bloodstream, and that this contributes to the distribution of CSFV in macrophages throughout the atretic follicles.     

Autoimmunity to a pathogenic retinal antigen begins as a balanced cytokine response that polarizes towards type 1 in a disease-susceptible and towards type 2 in a disease-resistant genotype.

Susceptible, but not resistant, strains of rodents immunized for induction of experimental autoimmune uveitis (EAU) with the uveitogenic protein interphotoreceptor retinoid-binding protein (IRBP) exhibit a type 1 response at the time of disease expression. Here we investigate the evolution of this response using the prototypic EAU-susceptible and EAU-resistant mouse strains, B10.A and BALB/c. Disease severity and IRBP-specific responses (proliferation, cytokines and antibody isotypes) were evaluated 7, 14 and 21 days after uveitogenic immunization. B10.A mice initially exhibited an IgG1-dominated antibody response, and their lymph node cells produced IL-4 and IL-5 in addition to IFN-gamma. On day 14 and 21, however, the IgG2a isotype became predominant, and the primed lymph node cells produced mainly IFN-gamma and IL-12. B10.A mice developed EAU before day 14. BALB/c mice initially produced IL-12 and IFN-gamma in addition to IL-5, IL-4 and IL-10. At later time points IL-12 and IFN-gamma production diminished, and IL-4, IL-5 and IL-10 increased. An IgG1-dominated antibody response was maintained throughout. BALB/c mice failed to develop EAU even at day 21. Thus, both susceptible and resistant genotypes initially mount a balanced, type 0-like cytokine response to a uveitogenic challenge, that subsequently polarizes towards type 1 in the susceptible strain and towards type 2 in the resistant strain.     

Different profiles of T-cell IFN-gamma and IL-12 in allergen-induced early and dual responders with asthma

BACKGROUND: IFN-gamma and IL-12 are anti-inflammatory cytokines released from various cells, including T cells. Allergen inhalation by atopic subjects with asthma results in 2 bronchoconstrictor phenotypes, termed isolated early and dual responders . Persistence of allergen-induced airway response and inflammation is a distinctive feature of dual responders. OBJECTIVE: To evaluate the roles of IFN-gamma and IL-12 in resolving allergen-induced airway inflammation by comparing T lymphocytes (CD4 + and CD8 + cells) producing these cytokines in isolated early and dual responders. METHODS: Twenty-four subjects with asthma (12 isolated early and 12 dual responders) were challenged with inhaled allergen. Peripheral blood and induced sputum were taken before and 1 day, 3 days, and 7 days after challenge. Frequency of IFN-gamma, IL-12, IL-4, and IL-13 producing CD4 + and CD8 + cells was assessed by using flow cytometry. RESULTS: After allergen, both CD4 + and CD8 + IFN-gamma positive cells in peripheral blood significantly decreased in dual responders only, whereas CD4 + and CD8 + IFN-gamma positive cells in induced sputum significantly increased in isolated early responders only. By contrast, IL-12 positive cells in peripheral blood significantly increased after allergen challenge only in isolated early responders. The ratio of CD4 + and CD8 + IL-4/IFN-gamma positive cells in peripheral blood significantly decreased in isolated early responders by 3 days and had recovered by 7 days. CONCLUSION: These results suggest that contrasting profiles of IFN-gamma and IL-12 production may be responsible for different time courses of allergen-induced airway responses between isolated early and dual responders.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus

Post-weaning multisystemic wasting syndrome (PMWS) has recently emerged as an important disease of pigs in North America, Europe and Asia. Porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) have been isolated from affected pigs. To investigate the pathogenicity of these isolates, groups of colostrum-deprived conventional pigs were inoculated with PCV2 alone (n=4), PPV alone (n=3) or dually with PCV2 and PPV (n=7) and examined post mortem between 21 and 26 days post-infection (dpi). Two control pigs were inoculated with an uninfected cell culture lysate. All pigs that received both viruses became dull at approximately 10-12 dpi and six of these animals subsequently developed jaundice. Hepatomegaly and enlarged kidneys were prominent post-mortem findings in these animals. Histopathological examination revealed severe macrophage infiltration, syncytia formation and numerous cytoplasmic and nuclear amphophilic inclusion bodies in lymphoid tissues. Granulomatous lesions were apparent in liver, lung, kidney, pancreas, myocardium, intestines, testis, brain and salivary, thyroid and adrenal glands. Abundant PCV2 antigen was detected in affected tissues. Only one of the four pigs inoculated with PCV2 alone developed clinical signs, but they all had histopathological lesions which, although less severe, were similar to those in the dually infected animals. The control pigs and those infected with PPV alone remained clinically normal and did not have gross lesions. The only histopathological lesion seen in these animals was mild interstitial nephritis in the pigs infected with PPV alone. These results indicate that lesions of PMWS can be induced by inoculating pigs with PCV2 alone, thereby fulfilling Koch's postulates. Concurrent infection with PPV increased the severity of the lesions, suggesting that co-factors are important in the pathogenesis of PMWS.