Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

Cross-reactivity between a monoclonal antibody that recognizes a tumor-associated antigen on bovine lymphosarcoma cells and blood lymphocytes from various mammalian species.

Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (MAB). By use of flow cytometry and radioimmunoprecipitation, 1 of the MAB (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (BL) from various mammalian species. By use of flow cytometry, the c143 MAB reacted with 10 to 49% of BL derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 MAB with BL from horses, pigs, human beings, and llamas ranged between 1:6.0 x 10(4) and 1:5.3 x 10(5); titer associated with BL of goats and sheep was 1:1.6 x 10(6); and that associated with BL of cattle was 1:4.3 x 10(7). The c143 MAB specifically immunoprecipitated 3 homologous proteins from cell extracts of caprine, ovine, and bovine BL (32-, 34-, and 36- to 37-kDa bovine proteins; 31-, 32-, and 36- to 37-kDa caprine proteins; and 31.5-, 33-, and 36- to 37-kDa ovine proteins), but none was immunoprecipitated from human, murine, canine, porcine, and llama BL. These results indicate that the avidity of the c143 MAB in binding to BL from ruminants (eg, goats, sheep, and cattle) is higher than that to BL from human beings, mice, dogs, horses, pigs, and llamas. In sheep, the c143 MAB could immunoprecipitate the aforementioned proteins from BL of the Suffolk breed, but not BL from the Corriedale breed, whereas the c143 MAB immunoprecipitated apparently identical proteins from BL of 4 breeds of cattle.     

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.     

Cloning of bovine MAIL and its mRNA expression in white blood cells of Holstein cows

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is known as an IkappaB protein induced after administration of bacterial lipopolysaccharide (LPS) to mice. In the present study, we cloned bovine MAIL cDNA and examined its mRNA expression in white blood cells isolated from Holstein cows. Bovine MAIL had more than 80% amino acid identities with murine and human MAILs, highly conserved ankyrin-repeat motifs and PEST-like sequences. Bovine MAIL mRNA was undetectable in isolated peripheral white blood cells, but rapidly induced (<1h) after stimulation by LPS and lipid A in vitro in a dose-dependent manner. The lipid A-induced MAIL mRNA expression was found in polymorphonuclear cells, monocytes/macrophages and total lymphocytes, but not in T-lymphocytes. MAIL mRNA was also induced in vivo in peripheral blood leukocytes of cows after intramammary injection of Escherichia coli derived from coliform mastitis. Thus, bovine MAIL, as rodent MAILs, is induced by inflammatory stimuli in specific immune cells in vitro and in vivo, suggesting a role in inflammatory responses to bacterial infection in cattle.