A comparison of different challenge methods for induction of atrophic rhinitis in pigs.

Transmission and development of atrophic rhinitis (AR) was studied in 5- to 15-week-old pigs (Groups 2-7) originating from a herd free of AR, and compared to unexposed healthy pigs (Group 1), and pigs from a herd with endemic AR (Group 8). At the start of the trial, pigs in Groups 2-5 were challenged intranasally twice a week for 3 weeks with pure cultures of bacteria originating from the endemic AR herd: Nontoxigenic Pasteurella multocida type A (PmA) plus Bordetella bronchiseptica phase I (Bb) (Group 2); PmA + toxigenic Pm type D (PmD) (Group 3); PmD only (Group 4); and PmD + Bb (Group 5). Group 6 pigs were challenged with nasal wash of pigs from the endemic AR herd, and Group 7 pigs were challenged by being housed together in the same pen with Group 8 pigs throughout the study. Nasal swabs of all pigs were cultured 5 times during the study. Serum was collected at 6 weeks post challenge. Average daily gain (ADG) and turbinate lesions (turbinate gross lesions by visual scoring and by Turbinate Perimeter Ratio, TPR, scoring, and histopathological lesions) were measured at the time of slaughter at 15 weeks of age. Mean TPR value for the Group 1 pigs was 1.64, which was significantly (P less than 0.05) different from the mean TPR value of 0.58 for the pigs from the endemic AR herd (Group 8), the 0.79 value for Group 6 pigs, and 1.03 value for Group 7 pigs. Of pigs challenged with pure bacterial cultures, only Group 5 (PmD + Bb) developed significant AR (mean TPR = 1.24). Only one pig in each of Groups 2 and 3, and two pigs in Group 4 showed TPR values indicative of AR (TPR less than 1.30). However, histopathological examination showed that those pigs were recovering from the infection 7 weeks post challenge. Constant exposure to certain bacteria or other factors in nasal washings, stress of crowding or poor environmental conditions might be required to experimentally produce AR in 5-week and older pigs similar to that in naturally infected pigs. There was no relationship between turbinate lesions and the isolation frequency or quantity of PmA, PmD, or Bb. Antibody levels against PmA or PmD had moderate to high correlation with TPR values (r = -0.694 and -0.503 respectively). ELISA values also corresponded well with the type of bacteria inoculated in each group of pigs and appeared to be a sensitive test for PmA, PmD, and Bb infections in pigs.(ABSTRACT TRUNCATED AT 400 WORDS)

Adherence of Bordetella bronchiseptica and Pasteurella multocida to swine nasal ciliated epithelial cells in vitro.

A bacterial adherence assay using swine nasal turbinate fragments was established. Turbinate fragments were incubated with Bordetella bronchiseptica or Pasteurella multocida type D at different concentrations or for different incubation times at 37 degrees C on a shaker at 120 rev/min. B. bronchiseptica phase I strains exhibited strong adherence to swine nasal ciliated epithelial cells. The number of adherent bacteria per cell increased when the bacterial concentration or incubation time increased (0, 15, 30, and 60 min); however, the number of adherent bacteria decreased after 3 or 6 hours' incubation due to the loss of cilia from cells. The optimal bacterial concentration and incubation time were 1 x 10(9) organisms/ml and one hour respectively, which resulted in 7.48 +/- 0.66 (Mean +/- SEM; B. bronchiseptica strain 03) and 9.31 +/- 0.54 (B. bronchiseptica strain 013) adherent bacteria per cell. In contrast to B. bronchiseptica phase I strains, rough phase strains of B. bronchiseptica and all P. multocida strains tested showed no adherence to swine nasal ciliated epithelial cells. All B. bronchiseptica phase I strains could agglutinate calf RBC but rough phase strains could not. Furthermore, pretreatment of B. bronchiseptica phase I organisms with 1 mg/ml or 2 mg/ml of trypsin significantly inhibited the adherence of B. bronchiseptica to ciliated epithelial cells; however, trypsin (2 mg/ml) treatment of bacteria did not decrease their ability to agglutinate calf RBC. From these results we conclude that, in addition to hemagglutinin, other proteinaceous components exist on the surface of virulent B. bronchiseptica that are sensitive to 2 mg/ml trypsin; these are suggested to be the adhesins for the adherence of B. bronchiseptica to swine nasal ciliated epithelial cells.

Inactivation of p16INK4a in primary tumors and cell lines of head and neck squamous cell carcinoma.

Inactivation of the p16INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5' CpG islands can serve as an alternative mechanism for the inactivation of the p16INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.

Flow cytometric analysis and chromosome sorting of barley (hordeum vulgare L).

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.

Modified alkali digestion method for pulmonary asbestos fibre counts.

In using the standard alkali digestion method for pulmonary asbestos fibre count, it was found that carbonaceous particles often obscured the presence of asbestos bodies (coated fibres) rendering their quantification inaccurate, particularly in lungs with a high soot particle content and a low fibre count. We applied the chloroform-ethanol separation technique from the bleaching agent digestion method to the alkali digestion method to remove the carbonaceous particles. It was found that coated fibres are much easier to visualize and count. The uncoated fibre count was nevertheless more accurate with the original method. The coated and uncoated fibre counts from 21 lung specimens were compared using both methods. The total fibre count was considered better derived from using both techniques.

Farrowing induction with a combination of prostaglandin F(2alpha) and a peripherally acting alpha(2)--adrenergic agonist AGN 190851 and a combination of prostaglandin F(2alpha) and oxytocin.

We studied the effect of prostaglandin (PG) F(2alpha)-AGN 190851 on farrowing induction and compared it with that of PGF(2alpha)-oxytocin. Eighty crossbred, multiparous sows were randomly assigned to the following 4 treatment groups of 20 sows each: 1) control, saline-saline; 2) PGF(2alpha) (10 mg/sow)-oxytocin (30 IU/sow); 3) PGF(2alpha) (10 mg/sow)-AGN 190851 (0.06 mg/kg); and 4) PGF(2alpha) (10 mg/sow)-AGN 190851 (0.1 mg/kg). Either PGF(2alpha) or saline was administered intramuscularly on Day 111 of gestation at 11:30 h; AGN 190851, oxytocin or saline was administered intramuscularly 20 h after the first injection. The PGF(2alpha)-AGN 190851 (0.1 mg/kg) treated sows had the shortest mean farrowing interval (2.1 +/- 1.6 h, mean +/- SD) compared with the remaining treatment groups (control: 67.1 +/- 26.2 h; PGF(2alpha)-oxytocin: 5.6 +/- 6.7 h; PGF(2alpha)-AGN 190851 [0.06 mg/kg]: 3.0 +/- 2.8 h). Duration of farrowing, litter size, litter weight and interval from weaning to first estrus in sows were not significantly changed by these treatments. The PGF(2alpha)-oxytocin group had a significantly higher stillbirth rate than the control group, whereas the PGF(2alpha)-AGN 190851 (0.1 mg/kg) group had the lowest number of pigs born dead and stillbirth rate among the 4 treatment groups. These results suggested that the PGF(2alpha)-AGN 190851 combination can be used as an alternative method to PGF(2alpha)-oxytocin for synchronizing farrowing.

Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.

Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.

Actinobacillus pleuropneumoniae culture supernatants interfere with killing of Pasteurella multocida by swine pulmonary alveolar macrophages.

The effect of Actinobacillus pleuropneumoniae culture supernatant on swine pulmonary alveolar macrophage (PAM) functions was studied. The A. pleuropneumoniae culture supernatant was toxic to PAMs when tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) release assays. Biological activity of the supernatant was ascribed to cytotoxins. Both the LDH and MTT assays were used for measurement of crude A. pleuropneumoniae cytotoxin concentration with good reproducibility. A preparation containing 6,800 toxic units/mL (determined by MTT assay) was used for subsequent experiments. The objective was to study the effect of crude cytotoxin on the ability of swine PAMs to kill Pasteurella multocida. Phagocytosis of opsonized P. multocida type A by PAMs was not efficient. Only 8% of incubated organisms were ingested by noncytotoxin-treated PAMs after 30 min phagocytosis. The bactericidal effect of noncytotoxin-treated PAMs only last for 60 min, after which, the rate of growth of surviving P. multocida exceeded the rate of bacterial killing by PAMs. Complete elimination of P. multocida by PAMs was not observed in this study. A total loss of ability to kill P. multocida by PAMs was seen when the PAMs were pretreated with a high concentration (340 toxic units/mL) of A. pleuropneumoniae cytotoxin. If the PAMs were pretreated with a low concentration (3.4 toxic units/mL) of cytotoxin, a significant reduction in the killing of P. multocida was still observed. The reductions in phagocytosis, phagosome-lysosome fusion (demonstrated using yeast particles of Candida albicans), and oxidative burst (demonstrated by nitro blue tetrazolium reduction (NBT) assay) may have contributed to the impaired killing of P. multocida by PAMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds.

An epidemiological study of porcine reproductive and respiratory syndrome (PRRS) within pig herds was conducted in 8 intensive farrow-to-finish pig farms. Persistence of PRRS virus (PRRSV) in pig herds was demonstrated by regular postmortem examination on 2 farms for a period of 2 y. Virus isolation and serum neutralization (SN) tests were performed on the sera collected from 9 groups of pigs (10 pigs/group) of various ages on 8 pig farms. Except for 1 farm, isolation rates of PRRSV reached the highest level of 70 to 100% of pigs 6 to 8 wk of age, which coincided with the lowest levels of maternal immunity. In 1 pig herd, sows (39 in total) with SN titers of < or = 1:2, 1:4-1:8, and > or = 1:16 were designated as groups 1, 2, and 3, respectively. Sera were obtained from their progeny (3 pigs randomly selected from each litter) at various ages from 0 to 22 weeks. A positive correlation (r = 0.377, P < 0.001) between the SN titers of sows and those of their progeny (1-week-old piglets) was observed. Pigs at the age of 6 wk, only 7.9% of group 1 pigs compared to 72.4% of group 3 pigs were seropositive. A significant difference (P < 0.01) in the percentage of pigs with PRRSV viremia among the 3 groups was observed, with the lowest level found in group 3 pigs. The isolation rates of PRRSV from serum reached the maximum at the age of 9 wk for all 3 groups. The results indicated that passively acquired serum antibodies conferred a protective effect for piglets; however, loss of passive immunity at various ages of pigs produced susceptible pigs that resulted in PRRSV persistence in the pig herds. Pigs 6 to 9 weeks old were the major reservoir for PRRSV in farrow-to-finish pig herds.

Epidemiological characteristics and financial costs of the 1997 foot-and-mouth disease epidemic in Taiwan.

Between March and July 1997, a devastating outbreak of foot-and-mouth disease (FMD), serotype O, occurred in pigs in Taiwan. A total of 6,147 pig farms with more than 4 million pigs were infected, and 37.7 per cent of the pigs in Taiwan either died (0.18 million pigs) or were killed (3.85 million pigs). The epidemic reached its peak during the fifth week after it was first recognised. During the eighth and ninth weeks, a two-dose blanket vaccination programme was instituted which led to a large reduction in new outbreaks. Except for two cities, the whole of Taiwan was declared an FMD-infected zone. During the four months in which new farm outbreaks occurred, 21.7 per cent of the pigs on infected farms showed clinical signs, and there was an overall mortality of 3.95 per cent. During the early stages of the epidemic, the incubation period was as short as 24 hours and the case fatality rates for suckling piglets reached 100 per cent. The financial cost of the epidemic was estimated at US$ 378.6 million, including indemnities, vaccines, carcase disposal plus environmental protection, miscellaneous expenses, and loss of market value. Owing to the ban on exports of pork to Japan, it is estimated that the total economic cost to Taiwan's pig industry will be about US$ 1.6 billion.

The ruptured abdominal aortic aneurysm--a diagnostic problem.

The ruptured abdominal aortic aneurysm continues to be a diagnostic problem. Review of 187 cases admitted to the Vancouver General Hospital showed that 92 cases were operated on. Of this surgical group, the diagnosis was correct in 61 and missed in 31 (34%). In the group which did not come to operation the condition was diagnosed correctly in 38, while in 57 (60%) it was completely unsuspected. The effects of early and late diagnosis and misdiagnosis were reflected in the increasing mortality rate of 46, 55 and 100% respectively. Means of improving the accuracy of diagnosis in this condition are discussed.

Efficacy of foot-and-mouth disease vaccine in pigs with single dose immunization.

Two experiments were conducted to demonstrate the efficacy of a commercial foot-and-mouth disease (FMD) vaccine in pigs born to well-vaccinated sows at various ages with a single injection under field conditions. The first experiment showed that single dose vaccination of pigs could be conducted at an age younger than 10 weeks. Second experiment demonstrated that pigs vaccinated once at the age of 8 weeks had mean serum neutralization (SN) titer of 1.89+/-0.95 log(10)SN(50) with full protection by challenge test at the age of 24 weeks. Results indicate that the most appropriate age for single dose FMD vaccination in pigs born to well-vaccinated sows would be at 8 weeks.

Hypermethylation of tumor-related genes in genitourinary cancer cell lines.

Hypermethylation of CpG island is a common mechanism for the inactivation of tumor-related genes. In the present study, we analyzed 13 genitourinary cancer cell lines for aberrant DNA methylation of 5 tumor-related genes using methylation- specific polymerase chain reaction (MSP). GSTP1 was methylated in 5 (38.5%), E-cadherin in 1 (8%), VHL in 1 (8%), and MGMT and hMLH1 in none (0%). Six out of thirteen genitourinary cancer cell lines had methylation of at least one of five genes; 5 had one gene methylated, and, 1 had two genes methylated. Methylation of these 5 genes was not detected in any of the bladder cancer cell lines. GSTP1 was methylated in all of the 3 prostate cancer cell lines. We conclude that aberrant hypermethylation may be an important mechanism for the inactivation of cancer-related genes in kidney and prostate cancer cell lines.