Detection of an isoform of alpha(1)-antitrypsin in serum samples from foals with gastric ulcers.

The objective of this study was to find serum indicators of gastric ulcers in foals. By using two-dimensional electrophoresis of serum proteins, three distinct spots were detected in samples from foals with gastric ulcers detected endoscopically. One of them appeared with high frequency and was identified by partial digestion with trypsin and subsequent nano-electrospray ionisation-tandem mass spectrometry (nanoesi-ms/ms) analysis as an alpha(1)-antitrypsin. Western blot analysis, using an antibody against human alpha(1)-antitrypsin, revealed at least two bands, of molecular weight 58 kDa and 55 kDa, in the sera. The 55 kDa band was detected in 44 of 47 serum samples from foals with gastric ulcers, but in only three of 22 serum samples from healthy foals.

Inhibition of pseudorabies virus replication by a dominant-negative mutant of early protein 0 expressed in a tetracycline-regulated system.

Pseudorabies virus (PRV) early protein 0 (EP0) consisting of 410 amino acids is a transactivator of viral genes. A mutant consisting of amino acids 1-113 exhibits dominant-negative properties. In order to assess the antiviral potential of the EP0 mutant, Vero cells were transformed with the EP0 mutant gene expressed in a tetracycline-regulated system. The transformed cell lines showed marked resistance to PRV infection when expression of the EP0 mutant gene was induced. In the transformed cell line infected with PRV, synthesis of the immediate-early protein (IE180) and of EP0 was inhibited, whereas the levels of IE and EP0 messenger RNA (mRNA) were not decreased, as compared with those of the control cell line. The present results suggest that the EP0 mutant may not alter the efficiency of the viral gene transcription but rather translation efficiency of the viral mRNA.

Suppression of pseudorabies virus replication by a mutant form of immediate-early protein IE180 repressing the viral gene transcription.

A mutant form of the immediate-early (IE) protein IE180 of pseudorabies virus (PRV), dIN454-C1081 is a strong repressor of the PRV IE gene promoter. In order to assess the antiviral potential of the IE180 mutant, HeLa cells were transformed with the mutant gene and then infected with PRV and herpes simplex virus type 1 (HSV-1). The transformed cell lines showed marked resistance to PRV infection, but were susceptible to infection with HSV-1, indicating that the IE180 mutant expressed in the stable cell line specifically inhibited PRV growth. In those cells infected with PRV, transcription of the PRV IE gene was repressed. In addition, the IE180 mutant exhibited a dominant-negative property in transient expression assay. The present results indicate that the resistance of the cells to PRV infection was due to repression of the IE gene transcription by the IE 180 mutant.

Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals.

The reliability of preparing bacteriological cultures from nasotracheal aspirates of foals routinely in order to diagnose R. equi pneumonia in foals was studied by isolating Rhodococcus equi from specimens obtained from 96 foals by nasotracheal aspiration with a silicon catheter. Results were compared with specimens obtained from 21 foals by transtracheal aspiration (percutaneous tracheal puncture). These 117 foals showed clinical signs of respiratory tract infection at sampling. R. equi was isolated from 14 of 21 (66.7%) specimens by transtracheal aspiration and from 59 of 96 (61.4%) specimens by nasotracheal aspiration, 649 of 655 isolates (99.1%) from the 73 positive specimens were virulent R. equi, and the culture-positive foals were diagnosed as having R. equi pneumonia. To assess the contamination of aspirates by organisms from the nasopharynx, the results of R. equi isolation from nasal swabs obtained from 56 of the 96 foals were compared to those obtained by nasotracheal aspiration from the same foals. R. equi was isolated from 2 of the 56 nasal swabs: one from a tracheal aspirate was positive, and the other was not. These results suggest that the nasotracheal aspiration technique, which is noninvasive and not associated with complications, could be used as an alternative to the transtracheal aspiration method, especially for the diagnosis of R. equi pneumonia in foals.

Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.

Physical and serologic examinations of foals at 30 and 45 days of age for early diagnosis of Rhodococcus equi infection on endemically infected farms.

OBJECTIVE: To evaluate results of physical and serologic examinations of foals at 30 and 45 days of age on 3 types of farms with various prevalences of clinical disease (endemic, sporadic, none) caused by Rhodococcus equi and to determine whether evaluations were helpful in early diagnosis and control of the disease. DESIGN: Prospective cohort study. ANIMALS: 144 foals at 30 and 45 days of age. PROCEDURE: During a 2-year period, 36 foals on farms at which R equi infection was endemic, 71 foals on farms at which the disease was sporadically detected, and 37 foals on farms without the disease were examined by means of auscultation of lungs, serum biochemical and hematologic analyses, and determination of antibody titers against R equi, using ELISA. Transtracheal aspirates were obtained from 14 of 32 foals that had clinical signs of disease and 7 of 41 seropositive foals that did not have clinical signs of disease. RESULTS: Prevalences of respiratory tract disease and seropositive conversion rates for 45-day-old foals on endemically and sporadically infected farms were significantly higher than on farms without the disease. Rhodococcus equi was isolated from tracheal aspirates of seropositive foals, even when clinical signs were not evident. CLINICAL IMPLICATIONS: Physical and serologic examinations of foals at 30 and 45 days of age were useful for early diagnosis of R equi infection, especially for foals on farms at which the disease was endemic.

Concentrations of IL-6 in serum and whey from healthy and mastitic cows.

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.

The first immunoglobulin-like domain of porcine nectin-1 is sufficient to confer resistance to pseudorabies virus infection in transgenic mice.

Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). Porcine nectin-1 mediates entry of pseudorabies virus (PRV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and bovine herpesvirus type 1 (BHV-1). The gD-binding domain of nectin-1 is the first or N-terminal immunoglobulin (Ig)-like domain of the entire ectodomain. Here, we generated three transgenic mouse lines expressing a fusion protein consisting of the first Ig-like domain of porcine nectin-1 and the Fc portion of porcine IgG1 to assess the antiviral potential of the first Ig-like domain of nectin-1 in vivo. All of the transgenic mouse lines showed significant resistance to PRV infection via intraperitoneal inoculation (survival rates of 67% to 100%). In the intranasal challenge, a lower but still significant protection was observed; 21% to 55% of the animals from the three transgenic mouse lines survived. The present results demonstrate that a soluble form of the first domain of porcine nectin-1 is able to exert a significant antiviral effect against pseudorabies virus infection.

A pan-specific promoter activity of the 213bp segment of the pseudorabies virus early protein 0 gene in transgenic mice.

Pseudorabies virus (PrV) early protein 0 (EP0) is a transactivator that plays important roles in the viral gene expression. To examine a promoter regulatory element for the EP0 gene expression in vivo, we have generated two transgenic mouse lines expressing the EP0 gene under the control of a 213bp 5'-flanking sequence of the EP0 gene. To analyze the tissue specificity of transgene expression, mRNA of the EP0 gene was monitored in various tissues from the transgenic mice by the reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. The EP0 gene expression was demonstrated in all tissues tested by the RT-PCR analysis. These tissues included skin, muscles (skeletal and heart), lung, liver, spleen, small intestine, kidney, brain and testis. These results indicated that the 213bp sequence of the 5'-flanking region of the EP0 gene is capable of driving expression of the EP0 gene in vivo and the promoter is pan-specific.

The pseudorabies virus immediate-early promoter directs neuronal tissue-specific expression in transgenic mice.

Pseudorabies virus (PRV) immediate-early (IE) gene product is required for expression of the viral early and late genes as a transactivator. The IE gene is expressed as the first gene among the viral genes after the infection. To examine the activity of the IE promoter in vivo, we have generated transgenic mice expressing transgenes under the control of the IE promoter. To analyze the tissue specificity of the transgene expression, mRNA of the transgene was monitored in various tissues from the transgenic mice by reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. A strong transgene expression was observed in the neuronal tissues by the RT-PCR analysis. These neuronal tissues included cerebrum, cerebellum and trigeminal nerve. Although the PCR product was hardly detected in other tissues by the RT-PCR analysis, specific PCR bands were detected in multiple organs (skin, skeletal muscles, heart muscles, lung, liver, spleen, small intestine and kidney) by Southern blot analysis using the RT-PCR products. These results indicate that although the IE promoter acts as a pan-specific promoter in vivo, it is capable of driving a high level of transgene expression in neuronal tissues.

Inhibition of pseudorabies virus replication by a chimeric trans-gene product repressing transcription of the immediate-early gene.

A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the immediate-early (IE) protein of pseudorabies virus (PRV) and a tail-truncated Vmw65 of herpes simplex virus 1, lacking the transcription activation domain, was constructed. The chimeric gene product inhibited transcription from the PRV IE promoter in a transient expression assay. A HeLa cell line stably transformed with the chimeric gene showed remarkable resistance to PRV infection. In the transformed cells infected with PRV, transcription of the PRV IE gene was repressed, indicating that the resistance of the cells to PRV infection was due to interference with IE gene transcription by the fusion protein.

Mapping of transcriptional regulatory domains of pseudorabies virus immediate-early protein.

The 180 kilodalton immediate-early protein (IE180) of pseudorabies virus functions as a strong transactivator of several different promoters and also as a repressor of its own transcription. To map the functional domains of IE180, we prepared various truncated mutants and analyzed their transcriptional regulatory activities using the chloramphenicol acetyl transferase (CAT) assay. Analysis of mutants truncated from the carboxy-terminal end of the 1,460-amino acid polypeptide showed that a polypeptide possessing amino acids 1 to 1,081 retained significant functions of transactivation and autoregulation potential. On the other hand, removing amino acids 1 to 131 resulted in a complete loss of transactivation potential, indicating that the domain responsible for transactivation is located in the amino-terminal end of IE180. Additional amino-terminal truncation up to amino acid 453 did not affect the autoregulation activity, indicating that the region between amino acids 454 and 1081 has autoregulation potential.

Mapping of a functional region conferring nuclear localization of pseudorabies virus immediate-early protein.

The immediate-early protein (IE180) of pseudorabies virus (PrV) is localized predominantly in the nuclei of infected cells. To define the nuclear localization signals within IE180, we prepared truncated mutants of IE180 and analyzed their localization in the transfected cells by indirect immunofluorescence. Analysis of mutants truncated from the carboxy-terminal end of the 1460-amino acid polypeptide showed that two regions including a short sequence of basic amino acid residues were associated with the nuclear localization of IE180. To assess whether these regions substantially function as signals for nuclear localization of the IE180 molecule, we then constructed two deletion mutants lacking each region. A mutant lacking amino acids 333 to 575 was detected in the nuclei of the transfected cells, whereas the other mutant lacking amino acids 900 to 950 was detected mainly in the cytoplasm. These results suggest that the region of amino acids 900 to 950 is responsible for nuclear localization of IE180.

Translational regulation of platelet-derived growth factor B chain (c-sis) mRNA by short open reading frames in the 5'-untranslated region.

Platelet-derived growth factor B chain (PDGF-B/c-SIS), the product of c-sis proto-oncogene, is a potent mitogen and chemoattractant for cells of mesenchymal origin. Expression of PDGF-B/c-SIS is regulated at the translational level, in addition to at the transcriptional level. The 5'-untranslated region (5'-UTR) of PDGF-B/c-sis mRNA is known to inhibit translation of the downstream coding sequences. The 5'-UTR contains putative influential elements, such as GC-rich elements, stem-loop structures and short open reading frames (SORFs). To clarify the inhibition mechanism of PDGF-B/c-sis mRNA translation, effects of three SORFs in the 5'-UTR on the translational regulation were investigated in transient expression assays. Introducing point mutation(s) in the initiation codons of SORFs affected the reporter gene expression in several cell lines (COS-1, U-2, JEG-3). Abrogation of three SORFs resulted in an increase of the reporter gene expression both in beta-galactosidase assay and Western blot analysis. These results suggest that SORFs in the 5'-UTR sequences have inhibitory effects on the translation of the downstream coding sequences.

Eimeria organisms develop in the epithelial cells of equine small intestine.

Histopathologic and immunohistochemical examinations were performed to determine the origin of host cells parasitized by Eimeria in the small intestines collected from five foals. Eimeria organisms at various stages (mainly microgametes and macrogametes) were frequently found in the cytoplasm of hypertrophied host cells in the lamina propria at the tips of villi of the jejunum and ileum. The cytoplasm of the host cell was immunohistochemically positive for cytokeratin AE1/AE3 and cytokeratin 13 and was negative for vimentin, desmin, alpha-smooth muscle actin, chromogranin A, neuron-specific enolase, and factor VIII. The host cells parasitized by Eimeria species had the immunostaining characteristics of epithelial cells but not of mesenchymal cells, endothelial cells of lacteals or capillaries, smooth muscle cells or neuroendocrine cells. These results suggest that the host cell of Eimeria species is possibly derived from intestinal epithelial cells and then displaced into the lamina propria of the small intestine.

Resistance to pseudorabies virus infection in transgenic mice expressing the chimeric transgene that represses the immediate-early gene transcription.

A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the immediate-early (IE) protein of pseudorabies virus (PRV) and a tail-truncated VP16 of herpes simplex virus 1, lacking the transcription activation domain, has been shown to repress transcription of the PRV IE gene, resulting in the inhibition of PRV growth in vitro. To assess the antiviral potential of the fusion protein in vivo, transgenic mice containing the chimeric gene under the control of the virus- and interferon-inducible Mx 1 promoter were generated. A transgenic mouse line showed marked resistance to PRV infection when the mice were challenged intranasally with PRV. Inhibition of PRV replication was also observed in monolayers of embryonic cells prepared from the transgenic mice. In the cells infected with PRV, transcription of the PRV IE gene was repressed. The present results indicate that the chimeric gene is able to exert a significant antiviral effect against PRV infection in vivo.