Polymorphisms of three neuroendocrine-correlated genes associated with growth and reproductive traits in the chicken.

The identification and utilization of potential candidate genes for QTL with significant effects on economically important traits are becoming increasingly important in poultry breeding programs. Chicken insulin-like growth factor binding protein 1 and 3 and signal transducers and activators of transcription 5B (STAT5B) genes are 3 essential nodes for signaling pathways and gene networks of growth and reproduction. The pooled DNA sequencing approach was used for identification of 9 SNP of the 5' upstream region of the 3 genes. A total of 826 individuals from Beijing You chicken were genotyped for 5 SNP using a modified PCR-RFLP method and the association with chicken growth and reproductive traits was studied using the GLM procedure. The T56039403C (T-808C) SNP of the insulin-like growth factor binding protein 1 gene was associated with BW at 10 wk of age (P = 0.0061), and the C56072547T (C-968T) SNP of the insulin-like growth factor binding protein 3 gene was associated with BW at 8 and 10 wk of age (P = 0.0056 and P = 0.0016, respectively). The C4535156T (C-1591T), G4533815A (G-250A), and G4533675C (G-110C) SNP of the STAT5B gene were associated with age at first egg (P = 0.0143, P = 0.0088, and P = 0.0114, respectively). Moreover, Lewontin's D' (|D'|) and r(2) of C4535156T and G4533815A SNP, C4535156T and G4533675C SNP, and G4533815A and G4533675C SNP of the STAT5B gene were 0.939 and 0.852, 0.967 and 0.858, and 0.971 and 0.896, respectively. The 3 SNP were strong-linked with each other and lay within a haplotype block. Our results suggest that these SNP were significantly associated with early growth or with sexual maturation in chickens, or both, and may be potential molecular markers for MAS.

Isolation of Salmonella enterica serotype choleraesuis resistant to ceftriaxone and ciprofloxacin.

Salmonella enterica serotype choleraesuis (S choleraesuis) usually causes systemic infections in man that need antimicrobial treatment. We isolated a strain of S choleraesuis that was resistant to ceftriaxone and ciprofloxacin from a patient with sepsis. Ciprofloxacin resistance was associated with mutations in gyrA and parC, whereas the ampC gene (bla(CMY-2)), responsible for ceftriaxone resistance, was carried by a transposon-like mobile element. This element was found inserted into finQ of a potentially transmissible 140 kb plasmid, with an 8 bp direct repeat flanking the junction regions. The appearance of this resistant S choleraesuis is a serious threat to public health, and thus constant surveillance is warranted.

Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis.

Salmonella enterica serovar Choleraesuis generally causes systemic human salmonellosis without diarrhea, and therefore, antimicrobial treatment is essential for such patients. The drug resistance information on this organism is thus of high value. Serovar Choleraesuis usually harbors a virulence plasmid (pSCV) of 50 kb in size. Of the 16 clinical isolates identified to be serovar Choleraesuis, all except one harbored a pSCV and seven of them carried a pSCV of more than 125 kb in size. A pSCV was defined as a plasmid carrying spvC and characteristic deletions detected by PCR and by DNA-DNA hybridization (for the former criterion). The results of PCR, restriction fragment profiles, and Southern DNA-DNA hybridizations of the profiles all indicated that such larger pSCVs were derived from the 50-kb plasmid recombined with non-pSCVs found in some clinical isolates. Fifteen of the 17 strains, including a laboratory strain, were then tested for drug resistance against 16 antibiotics with E-test and the dilution method. The laboratory strain, which harbored a 50-kb pSCV and a 6-kb non-pSCV, was resistant only to sulfonamides (SUL), and its resistance gene, sulII, checked with PCR and DNA-DNA hybridization, was located on the 6-kb non-pSCV. All 14 clinical strains were resistant to multiple drugs. Of the 14, 7 were resistant to SUL, and the resistance gene was located on a plasmid. The sulII gene, but not bla(TEM-1), was carried only on the 6-kb non-pSCV. Of the remaining six large plasmids, three of 90 kb, two of 136 kb, and one of 140 kb, the last three were pSCVs and carried the other SUL gene (sulI) and the bla(TEM-1) gene. The six strains were also resistant to trimethoprim-sulfamethoxazole. None of the 50-kb pSCVs carried resistance genes. These drug resistance genes on the large pSCVs were apparently also acquired through recombination.

Lack of evidence of an association between the carriage of virulence plasmid and the bacteremia of Salmonella typhimurium in humans.

The involvement of the virulence plasmid (pSTV) of Salmonella typhimurium in human salmonellosis was examined. Most of the 224 clinical strains isolated from the blood (53) and nonblood samples (171) contained a 90 kb or larger plasmid, most of which were pSTV. The rates of pSTV carriage in the isolates showed no statistically significant difference between those derived from the blood and those from other sources (87% vs. 83%; chi2=0.49, 0.1

Typhoid fever in children: a fourteen-year experience.

From 1982 to 1995, 71 children admitted in our medical center were diagnosed to have typhoid fever by culture or serology. Of the 71 children, most (83%) were aged 5-15 years. These children usually presented with fever and gastrointestinal symptoms, including abdominal pain, diarrhea, nausea or vomiting, and constipation. Hepatosplenomegaly was the most common physical sign observed and abdominal tenderness ranked the second. Thrombocytopenia occurring in 9 patients (13%) was the most common mode of complication. Other complications included intestinal perforation (3%), rectal bleeding (3%), ascites or pleural effusion (4%), and meningitis (1%). The incidence of complications tended to be higher among children 5 years of age or older (p = 0.31). Most patients responded well to appropriate antimicrobial therapies. There was no mortality. Relapse was observed in two children, although both had received 10 days of chloramphenicol therapy. The clinical isolates of Salmonella typhi were susceptible in vitro to all the antibiotics tested, including chloramphenicol, which, however, showed a higher MIC90 level than other drugs tested. In conclusion, there were age-specific differences of typhoid fever in children in terms of the incidence and morbidity and antibiotic resistance of S. typhi has not been a problem in this area at least up to 1995.

Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection.

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine. Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.

Prevalence of the virulence plasmids of nontyphoid Salmonella in the serovars isolated from humans and their association with bacteremia.

To determine if the virulence plasmid is one of the elements contributing to Salmonella bacteremia in humans, 436 clinical Salmonella isolates of different serovars were examined by a specific multiplex polymerase chain reaction assay for the presence of a virulence plasmid. These serovars showed differences in their ability to produce particular disease syndrome in humans. In the serovars usually causing bacteremia without concomitant gastroenteritis (primary bacteremia), i.e., S. choleraesuis, S. dublin, and S. enteritidis in this study, the rate of virulence plasmid carriage was 100%, while among those occasionally generating bacteremia following an episode of gastroenteritis (secondary bacteremia), the majority were plasmidless. Only a portion of S. typhimurium strains harbored a virulence plasmid; however, the rates of virulence plasmid carriage in S. typhimurium were not statistically different between non-fecal and fecal isolates (90% vs. 85%, 0.1 < P < 0.9). These results indicate that the virulence plasmids may be important for primary bacteremia, but not secondary bacteremia, to occur.

A clinical trial comparing oral azithromycin, cefixime and no antibiotics in the treatment of acute uncomplicated Salmonella enteritis in children.

OBJECTIVE: The objective of this study was to perform a prospective, randomized, controlled study to evaluate the role of azithromycin and cefixime in the treatment of uncomplicated non-typhoid Salmonella enteritis in children. METHODOLOGY: Patients with Salmonella enteritis were randomized to receive oral azithromycin (10 mg/kg/day once daily), cefixime (10 mg/kg/day divided twice daily) or no antibiotics for 5 days. The patients were followed up for the duration of their symptoms. Stool samples were sent for culture weekly following the therapy until two consecutive negative results were obtained. Susceptibility of the isolates to antibiotics was tested by the disk diffusion method. RESULTS: Forty-two patients with acute, uncomplicated, culture-confirmed Salmonella enteritis were studied. Duration of diarrhoea and time to defervescence after the therapy were not significantly different for patients treated with azithromycin, cefixime, or no antibiotics; there also were no significant differences with respect to the rate of clearance of Salmonella from stools among the three groups. Salmonella typhimurium was the most common serotype isolated. All 42 isolates were sensitive to cefixime, while two strains (5%) were resistant to azithromycin. CONCLUSION: Azithromycin or cefixime provides no benefit to paediatric patient with uncomplicated Salmonella enteritis.

In vitro evaluation of intracellular activity of antibiotics against non-typhoid Salmonella.

Non-typhoid salmonellae are the most common causative organisms of bacterial enteritis in children. Clinical studies have failed to show any influence of various antibiotics on the natural course of acute salmonella enteritis. Poor penetration of antibiotics into phagocytic cells that contain intracellular Salmonella spp., and possible intracellular antibiotic inactivation have been considered as possible reasons for this. In this study, we used an in vitro model to assess the intracellular activity of antibiotics against non-typhoid salmonellae. The survival of intracellular Salmonella spp. in P388D1 cells, a mouse macrophage cell line was measured in the presence of various antibiotics. Except for gentamicin, which entered phagocytes poorly, ofloxacin, azithromycin, chloramphenicol and three beta-lactam antibiotics, ampicillin, cefixime and ceftriaxone, exhibited bacteriostatic activity against susceptible intracellular Salmonella spp. at an extracellular concentration equal to the minimal inhibitory concentration (MIC). At a concentration of 10 x MIC, neither chloramphenicol nor the three beta-lactam antibiotics produced a bactericidal response; however, both ofloxacin and azithromycin were bactericidal after 8-24 h of incubation. The results showed that fluoroquinolones and new macrolides were more efficient than the other antibiotics in eradicating intracellular salmonella and might be useful agents for the treatment of non-typhoid salmonella enteritis in children. Clinical trials should be considered.

Comparative physical and genetic maps of the virulence plasmids of Salmonella enterica serovars typhimurium, enteritidis, choleraesuis, and dublin.

Using fragment profiling, PCR, and Southern hybridization, we found that Salmonella enterica serovar Choleraesuis harbored virulence plasmids of various sizes, whereas serovars Typhimurium, Enteritidis, and Dublin carried a plasmid of a unique size. Also, the virulence plasmid of Typhimurium contained genes in the same order detected in the other three plasmids, all of which contained deletions.

Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid.

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to human salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.

Predictors for extraintestinal infection of non-typhoidal Salmonella in patients without AIDS.

To identify the risks and predictors for extraintestinal infection (EII) in patients with non-typhoidal salmonellosis, we undertook a study of 398 patients with cultures positive for non-typhoidal Salmonella seen at Chang Gung Memorial Hospital and Chang Gung Children's Hospital between November 1993 and October 1994. Salmonella choleraesuis was the most invasive serotype observed. S. panama, S. typhimurium and S. schwarzengrund were the commonest causes of EII among those serotypes usually causing gastroenteritis. Pre-existing underlying disease, particularly immunosuppressive disease, was the most important risk factor that may have predisposed adult patients to have EII. Old age (> or = 60 years) and isolation of invasive serotypes were also frequently associated with EII in adult patients. The characteristics of paediatric patients with a high probability of having EII were: < 3 years of age, abnormal blood test results (a leucocyte count > or = 15,000/mm3 or < 5000/mm3, immature leucocytes > or = 10% of total leucocytes, and a C-reactive protein concentration > or = 50 mg/l); and isolation of invasive serotypes. This information can be an aid to early diagnosis and treatment of EII caused by non-typhoidal Salmonella.

Frequency of Epstein-Barr virus-associated gastric adenocarcinoma in Taiwan.

Eighty-two gastric adenocarcinomas (32 intestinal and 50 diffuse type) were investigated for the presence of Epstein-Barr virus (EBV) DNA by amplifying the 78-bp fragment from the EBNA1 gene with polymerase chain reaction. EBV was detected in 17 (20.7%) of the 82 gastric tumorous specimens. Of these 17 EBV-positive cases, only one was EBV-positive in the adjacent non-tumorous tissue. EBV-positive gastric adenocarcinoma was present in 15.6 and 24.0% of the intestinal and diffuse type tumors, respectively, but within the two classes there was significant non-homogeneity by type. EBV was found more frequently in adenocarcinomas of the tubular (25%) and poorly differentiated (30.3%) types. EBV involvement was found more in male than in female patients. Eighty of the 82 tumors were also checked for the prevalence of p53 gene mutation. Of the 17 EBV-positive gastric adenocarcinomas, only two showed p53 gene mutations. The p53 mutation rate was lower in EBV-positive tumors (11.8%) than in EBV-negative tumors (25.4%).

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells.

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

A pilot study of seven days of ceftriaxone therapy for children with Salmonella enterocolitis.

BACKGROUND: The most effective therapy for non-typhoid Salmonella enterocolitis is still unknown. Traditionally, unless extraintestinal complications are present, antimicrobial drugs are not recommended, since earlier trials have shown that antibiotics such as ampicillin, chloramphenicol, or co-trimoxazole, do not shorten the duration of diarrhea and may even prolong convalescent fecal carriage of the bacteria. However, the recently-developed third generation cephalosporin ceftriaxone has been used successfully in the treatment of typhoid fever and other systemic salmonellosis. A controlled, pilot study was therefore undertaken to evaluate the efficacy of intravenous ceftriaxone in the treatment of children with non-typhoid Salmonella enterocolitis. METHODS: Fifteen children with Salmonella enterocolitis and bacteremia who were eligible for antibiotic therapy were given ceftriaxone intravenously for 7 days and 15 children with enterocolitis but without bacteremia who were admitted for supportive treatment during the study period were selected as the control group. Available stool samples collected on days, 7, 14, and 30 after the completion of the drug therapy were checked for the presence of the bacteria using polymerase chain reaction (PCR) and culture methods. RESULTS: The result showed that the duration of diarrhea was not significantly affected by ceftriaxone treatment. However, the difference in the rate of clearance of Salmonella from stools, as defined by negative stool cultures and PCR, was statistically significant between the two groups on posttreatment days 7 and 14. Only one patient given ceftriaxone was shown to have recrudescence of the bacteria in feces on day 14. One month after therapy, PCR was positive in two of the ten cases tested and one of these two experienced a relapse of diarrhea, whereas bacterial carriage was maintained in 63% of the control patients. CONCLUSION: A prompt eradication of Salmonella in feces was observed in most of the patients treated with ceftriaxone in this study. If further studies confirm the efficacy of this therapy and the risk of inducing drug resistance is minimal, the epidemiologic problem created by convalescent fecal bacterial carriage may justify a short-course of ceftriaxone therapy for children with Salmonella enterocolitis.

Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.

Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rat.

The mitogenic effect of activated coagulation factor X (factor Xa) was examined in cultured aortic smooth muscle cells (VSMC) from Wistar-Kyoto rats (WKY). Factor Xa stimulated DNA synthesis and cell growth in VSMC, not through the phospholipase C-protein kinase C pathway because increase of inositol monophosphate (IP) accumulation and intracellular Ca2+ concentration was not observed, but probably via the PDGF receptor tyrosine kinase pathway since the pathway's components, Ras, Raf-1, MAPK (both 42 and 44 kD), and the transcription factors, c-Fos and c-Jun, were activated. These appeared to be effected by the serine protease activity of factor Xa, since in the presence of serine protease inhibitors such as PMSF, leupeptin, benzamidine, TAP anticoagulant, and TLCK, the latter three being specific inhibitors of the factor Xa, active site, the effects were completely blocked. Anti-factor Xa mAb, 5224, which specifically negated the activity of factor Xa, also inhibited completely the mitogenic effect of factor Xa, but not that of thrombin. Addition of PDGF did not affect the effect of factor Xa, which, however, was inhibited by anti-PDGF-AB antibody. This observation and the activation of PDGF receptor tyrosine kinase pathway suggested that the factor Xa might exert its effect via PDGF-like function. Direct measurement confirmed that factor Xa stimulated the release of PDGF from VSMC. Factor Xa, therefore, exerts serine protease activity on VSMC, causing somehow the release of PDGF, that in turn acts on the PDGF receptor tyrosine kinase; the pathway is then turned on, leading eventually to DNA synthesis and cell proliferation.