Low expression of AcrB in the deoxycholate-sensitive strains of Salmonella enterica subspecies enterica serovar Pullorum.

We investigated the mechanism responsible for bile susceptibility in three deoxycholate-sensitive (DCs) strains of Salmonella enterica subspecies enterica serovar Pullorum isolated in 1958 in Japan. Of the genes encoding the AcrAB-TolC efflux system, the expression of acrB mRNA was 10-fold lower in the DCs strains than in a deoxycholate-resistant (DCr) strain, whereas those of the acrA and tolC genes were two-fold lower. These results suggested that low expression of acrB was strongly correlated with bile susceptibility in the DCs strains. In addition, the increase in tolC expression levels was not detected in the DCr mutants derived from the DCs strains, suggesting that difference in the expression levels of tolC is not associated with bile susceptibility.

The effect of feeding lactic acid to Salmonella typhimurium experimentally infected swine.

Antimicrobial resistant Salmonella are becoming more prevalent. Therefore, alternative methods to control swine Salmonella infection must be explored. We examined whether feeding lactic acid to swine is an effective way to control clinical and subclinical Salmonella Typhimurium infection in these animals. In this experiment, swine were inoculated with 5.6 x 10(7) CFU (hi-ST) or 5.6 x 10(5) CFU (lo-ST) of S. Typhimurium per swine to reproduce clinical and subclinical infection. The swine were either fed a commercial feed supplemented with 2.8% lactic acid (LA) or the commercial feed without supplementation (C) to examine the effect of feeding lactic acid. Twenty 21 and 22 days old swine were divided into 4 groups, LA-hiST, C-hiST, LA-loST and C-loST, and fed the respective feed. They were inoculated S. Typhimurium at 51 and 52 days old. Clinical symptoms and the number of S. Typhimurium shed in feces were evaluated. The LA-hiST group did not show obvious clinical symptoms, such as diarrhea or febrile response, but the C-hiST group did show clinical symptoms. The number of S. Typhimurium shed in the feces of the LA-hiST group was lower than in that of the C-hiST group, and that of the LA-loST group was lower than that of the C-loST group. Our data suggest that dietary supplementation with 2.8% lactic acid can be an effective way to control clinical and subclinical infections of S. Typhimurium in swine.

Gross thymic extract, Thymax, induces apoptosis in human breast cancer cells in vitro through the mitochondrial pathway.

Previous studies have shown that thymic extracts possess antitumor and antimetastatic properties, but the mechanisms are not completely understood. Therefore, in this study the ability of the gross thymic extract Thymax to induce apoptosis in human breast cancer cell line (MCF-7) cells in vitro was evaluated. Tumor cells were cultured with different concentrations of Thymax for 24 h and the apoptotic response was assessed by propidium iodide and TUNEL assays. Activation of caspases and changes in mitochondrial membrane potential (MMP) were monitored by flow cytometry and the expression of Bcl-2 and Bax was determined by Western blot analysis. Thymax induced apoptosis in monolayer MCF-7 cells in a dose-dependent manner; at concentrations of 2.5, 5 and 10% (v/v) it caused 9%, 10% and 25% apoptosis, respectively, as compared to 6% for control cancer cells without treatment. The induction of apoptosis by Thymax was associated with activation of caspases 8 and 9, and the addition of a pan caspase inhibitor partially inhibited Thymax-induced apoptosis by 20%. In addition, the MMP was decreased significantly at Thymax concentrations of 5%-20%, which was associated with a decrease in the protein expression of Bcl-2 and an increase in Bax. These results suggest that Thymax exerts its effects via the mitochondrial pathway of apoptosis and may represent a new class of adjuvants for the treatment of breast cancer.

Reproduction of adenoviral gizzard erosion by the horizontal transmission of fowl adenovirus serotype 1.

The horizontal transmission ability of fowl adenovirus (FAV) serotype 1 99ZH strain, isolated from chickens exhibiting gizzard erosion, was investigated. Twelve 13-day-old specific pathogen-free chickens were inoculated orally with 10(6) TCID(50)/0.05 ml of the strain. An in-pen contact group (chickens in the same pen with inoculated chickens), hedge contact group (chickens in a pen connected with pens housing inoculated chickens), non-contact group (chickens in a separate pen placed at a distance of 70 cm from the connected pens), human exposure group (chickens in the next room and attended last every day) and negative control group were examined. Each group consisted of 11 or 12 uninoculated chickens. Gizzard lesions were grossly or histologically observed from 10 days after exposure (DAE) in the in-pen contact group, and from 15 DAE in the hedge contact and non-contact groups. The FAV gene was detected by polymerase chain reaction performed on cloacal swabs taken on 5 and 13 DAE from chickens in both contact groups, and on 20 and 26 DAE from those in the non-contact group. Serum neutralizing antibodies against FAV serotype 1 were detected in chickens from 13 and 26 DAE in both contact groups and in the non-contact group, respectively. In the human exposure and negative control groups, no infection was observed. We conclude that FAV-99ZH strain spreads rapidly through direct contact with inoculated chickens, and slowly through non-contact transmission, and that adenoviral gizzard erosion is reproduced by this horizontal transmission.

Long-term prevalence of antimicrobial-resistant Salmonella enterica subspecies enterica Serovar infantis in the broiler chicken industry in Japan.

Eleven broiler isolates of Salmonella Infantis obtained between 1989 and 1998 were examined for antimicrobial susceptibility and pulse field gel electrophoresis (PFGE) profiles. Seven strains of S. Infantis isolated after 1993 harbored similar antimicrobial susceptibilities to the recent isolates between 2001 and 2003. In comparison of PFGE profile with 22 isolates obtained from 22 apparently healthy broiler chickens between 2001 and 2003, the predominant cluster included the seven strains isolated after 1993. We could not clarify the reasons why the serovar has been prevalent in the broiler industry for a long time, but current antimicrobial usage is not always linked to its prevalence.

Comparison of the polymerase chain reaction-restriction fragment length polymorphism pattern of the fiber gene and pathogenicity of serotype-1 fowl adenovirus isolates from gizzard erosions and from feces of clinically healthy chickens in Japan.

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.

Prevalence of the virulence plasmid in Salmonella Typhimurium isolates from pigs.

To determine the prevalence of the virulence plasmid in Salmonella Typhimurium isolates from pigs in Japan, a total of 106 porcine isolates were subjected to PCR amplification for the detection of the virulence plasmid. Out of the isolates of S. Typhimurium, 38 (35.8%) harbored the virulence plasmid. The presence of the virulence plasmid was widely observed in the isolates from systemically infected pigs (92.0%, 23/25), compared with diarrheic (18.8%, 12/64) and apparently healthy pigs (17.6%, 3/17) (P<0.01).

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.

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.

An epizootic of subcutaneous tumors associated with subgroup A avian leukosis/sarcoma virus in young layer chickens.

An outbreak of subcutaneous tumors in young layer chickens in a flock in Japan was investigated. Tumors appeared as extensive swelling or bulbous protrusions of the integument and were observed in the head or wing of chickens approximately 9 wk old, with a prevalence of 0.4% (157 of 42,000) in the affected flock. Histologically, two types of tumor were observed: myxoma containing abundant hyaluronic acid and neurofibroma with hyperplasia of the Herbst corpuscles. Ultrastructurally, type C retroviruses, such as viral particles, were found in the tumors. The tumors were specifically stained by immunohistochemistry using monoclonal antibodies against the subgroup A avian leukosis/sarcoma virus (ALSV) and yielded a positive reaction to primers specific for subgroup A ALSV by reverse transcriptase-polymerase chain reaction assay. The virus was isolated from the tumors. Seventeen of 20 clinically normal chickens in the affected flock showed antibodies against ALSV. These results suggest that subcutaneous tumors are associated with subgroup A ALSV infection.

Pathogenicity of serotype 8 fowl adenovirus isolated from gizzard erosions of slaughtered broiler chickens.

The pathogenicity of serotype 8 fowl adenovirus (FAV), isolated from gizzard erosions of slaughtered broiler chickens, was investigated. In experiment 1, 29 5-day-old specific-pathogen-free (SPF) chickens were inoculated with the isolates of serotype 8 FAV, M013 (group 1) or G0054 (group 2) strain, via an oral route. There were no clinical signs in any of chickens after inoculation, and mild gizzard erosions were observed macroscopically and microscopically in three inoculated chickens of group 2. FAV was recovered from gizzards and rectums but was not recovered from pancreas and livers from chickens in both inoculated groups. In experiment 2, 27 1-day-old SPF chickens were inoculated with the G0054 strain by intramuscular route. Five, 6, and 3 inoculated chickens died on days 3, 4, and 5 postinoculation (PI), respectively. Four, 3, 1, and 1 inoculated chickens became moribund with severe clinical signs such as ruffled feathers, severe depression and closed eyes from days 3 to 6 PI, respectively. Macroscopically, the common characteristic of the gross lesions of dead chickens and euthanized moribund chickens was discoloration of liver. FAV was recovered from the gizzard, liver, pancreas and rectum. Virus titers in the liver and pancreas were high until day 6 PI. Histologically, necrotizing hepatitis and pancreatitis with intranuclear inclusion bodies were observed in the inoculated chickens. These results indicate that some strains of serotype 8 FAV are able to reproduce not only gizzard erosion by oral inoculation but inclusion body hepatitis (IBH) by intramuscular inoculation.

Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs.

The growth of Salmonella Choleraesuis was examined in Rappaport Vassiliadis broth (RV) and Hajna-tetrathionate broth (HTT) at 37 and 42 degrees C. As the enrichment in RV at 37 degrees C was satisfactory for isolating S. Choleraesuis, we used this enrichment for isolation from the samples collected from 15 asymptomatic pigs reared on a S. Choleraesuis contaminated farm. S. Choleraesuis was frequently isolated from six pigs (40.0%) under field conditions. The isolation of other Salmonella serovars than S. Choleraesuis was attempted by using both RV enrichment at 37 degrees C and HTT enrichment at 42 degrees C. Salmonella organisms were isolated from 156 (44.8%) of 348 fecal samples and more frequently with HTT at 42 degrees C (43.4%) than with RV at 37 degrees C (20.9%). If other serovars in addition to S. Choleraesuis are to be surveyed, HTT enrichment should be used in combination with RV enrichment.

Isolation and serological survey of Salmonella in pigs in Japan.

A total of 267 fecal and serum samples collected from individual pigs reared on a Salmonella-positive farm were subjected to bacteriological and serological examinations of Salmonella. Salmonella was isolated from 47 pigs (17.6%) and prevalence of antibody to lipopolysaccharide (LPS) of S. Typhimurium, which was partly common to S. O4, 12: d: -, was observed in 90 pigs (33.7%). Salmonella was isolated from 26 (28.9%) of 90 antibody-positive pigs and 21 (11.9%) of 177 antibody-negative pigs. Twenty-one of 36 pigs (58.3%) positive for S. O4, 12: d: -, five of 10 pigs (50.0%) positive for S. Havana, and none for S. Anatum had antibodies. Thus, seropositive rates were higher than isolation-positive rates, and antibody prevalence was associated with serovars of the isolates. Then, we analyzed antibody prevalence among pigs on Japanese pig farms. The antibodies to LPS of S. Typhimurium were found in 195 of 1,498 pigs (13.0%) and in at least one serum sample on 35 of 52 farms (67.3%). Our results indicate that Salmonella does not seem to be so prevalent in pigs though it is widely prevalent among pig farms.

Isolation of Salmonella from diarrheic feces of pigs.

A survey of Salmonella was carried out in fecal samples of 887 pigs with diarrhea collected from 235 pig farms between April 1996 and March 2001. Salmonella was isolated from 84 feces (9.5%) of 887 pigs and from 45 (19.1%) of 235 farms. The higher prevalence was found in weaned pigs (12.4%) and fattening pigs (17.3%) than in sows (4.2%) and suckling pigs (4.5%). Isolation rates of S. Typhimurium were higher from weaned and fattening pigs than from the others. Therefore, risk of horizontal infection of S. Typhimurium will increase, if no adequate health managements are practiced when weaned and fattening pigs have diarrhea.