Serotype Diversity and Antimicrobial Resistance among Salmonella enterica Isolates from Patients at an Equine Referral Hospital.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most (n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as blaCMY-2, blaSHV-12, blaCTX-M-27, and blaTEM-1B, were detected. The qnrB2 and aac(6')-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3')-Ia, aac(6')-IIc), streptomycin (strA and strB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) and tet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Identification of a Plasmid-Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples.

A recent increase in plasmid-mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti-microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole-genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid-mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks.

Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 µs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Hookworm-related cutaneous larva migrans in patients living in an endemic community in Brazil: immunological patterns before and after ivermectin treatmen.

Hookworm-related cutaneous larva migrans (Hr-CLM) is caused by animal hookworm larvae migrating in the human epidermis where they elicit an inflammatory response. This study describes the immunological profile in Hr-CLM patients. In 77 Hr-CLM patients from Manaus, Brazil, peripheral eosinophils were counted, and serum concentrations of total immunoglobulin E (IgE) and selected cytokines were determined by ELISA before and after treatment with ivermectin. Controls included patients' household members (endemic controls), non-endemic Brazilian and Japanese individuals. Eosinophil counts and total IgE in Hr-CLM patients were higher than in controls and correlated with disease severity. Concentrations of interleukin (IL)-4, IL-5, IL-6, and IL-10 were higher in Hr-CLM patients than in endemic controls (p < 0.001) while no differences were detected for interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-1ß, IL-2, or transforming growth factor (TGF)-ß. Following ivermectin treatment, numbers of eosinophils and concentrations of IL-4, IL-5, and IL-10 decreased whereas IgE, IFN-γ, and TGF-ß concentrations increased. The IL-5/IFN-γ ratio declined from 5.9 (interquartile range [IQR] 0.8-31.6) before to 0.1 (IQR 0.05-0.2; p = 0.001) after treatment. Thus, although an impact of other infections on the immune parameters determined cannot be excluded, Hr-CLM in endemic areas is associated with eosinophilia and elevated cytokine levels, particularly of IL-5 and IL-10, which decrease following ivermectin treatment.

Contamination, distribution and pathogenicity of Toxocara canis and T. cati eggs from sandpits in Tokyo, Japan.

The contamination, distribution and pathogenicity of Toxocara canis and T. cati eggs in sandpits in the Tokyo metropolitan area, Japan, are described. A total of 34 sandpits were examined, 14 of which were contaminated with T. cati eggs, as assessed by the floatation method and polymerase chain reaction (PCR) analysis. Two naturally contaminated sandpits were investigated to determine the vertical and horizontal distribution of eggs, and an inverse relationship between the sand depth and number of eggs was observed. To examine the pathogenicity of the eggs, three ICR mice were inoculated with 300 eggs, which were recovered from sandpits. The mice exhibited eosinophilia in the peripheral blood and IgG antibody production in the sera after 3 weeks of infection. Most migrating larvae were recovered from carcasses, although three were found in the brains of two infected mice. These three larvae were determined to be T. canis by PCR, revealing that not only T. cati, but also T. canis eggs could be found in sandpits and, further, that eggs recovered from sandpits have the ability to invade a paratenic host.

Transfer of blood urea nitrogen to cecal microbial nitrogen is increased by mannitol feeding in growing rabbits fed timothy hay diet.

The presence of the fermentable sugar d-mannitol in the diet improves nitrogen (N) utilization in rabbits. To clarify the mechanism by which d-mannitol improves N utilization, we studied the effect of d-mannitol on the fate of blood urea N in growing rabbits. Growing rabbits received a control diet or a diet containing d-mannitol, which were formulated by adding 80 g/kg glucose or d-mannitol to timothy hay. After 9 days of feeding of the experimental diets, 15N-urea was administrated intravenously under anesthesia 1 h before slaughter. The blood urea level (concentration of both urea N (43.6% of the control group (CG), P < 0.05) and 15N (95% of the CG, P < 0.05) in blood serum) was reduced in the mannitol group. The concentration and amount of N, and 15N atom % excess in the contents of the cecum and colon were higher (P < 0.05) in the rabbits fed the mannitol diet than in rabbits fed the control diet, especially in the cecum. The consumption of mannitol caused bacterial proliferation in the cecum characterized by marked short-chain fatty acid production (165% of the CG, P < 0.05), decreased cecal ammonia N (73% of the CG, P < 0.05) and elevated cecal bacterial N (150% of the CG, P < 0.05). On the other hand, addition of d-mannitol to the diet decreased N (80% of the CG, P < 0.05) and 15N (77% of the CG, P < 0.05) excretion in the urine. These results indicate that d-mannitol increases the transfer of blood urea N to the large intestine, where it is used for bacterial N synthesis.

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples.

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.

Formation mechanism studies of phenylene-bridged periodic mesoporous organosilicas (PMOs).

The formation of phenylene-bridged periodic mesoporous organosilicas (PMOs) in the presence of three diblock copolymers differing in hydrophobic hydrocarbon chain lengths was investigated. Hexaethylene glycol octadecylether (C(18)(EO)(6)), hexaethylene glycol hexadecylether (C(16)(EO)(6)), and hexaethylene glycol dodecylether (C(12)(EO)(6)) were chosen in order to obtain insight on the influence of the hydrocarbon chain length on the mesostructure. 1,4-Bis(triethoxysilyl)benzene (BTEB) was used as organosilica precursor under mild acidic conditions. The reactions were followed by in situ small-angle X-ray diffraction (SAXD) on-time in a capillary flow setup. It was found that during the reaction the formation of different structures was observed, which is ascribed to the hydrolysis and condensation of the organosilica precursor. In all cases, different structures evolve with time and phase transitions are observed during the measurements independent of the hydrocarbon chain length.

Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction.

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

An improved method for recovery of muscle-stage larvae from mice infected with Toxocara canis.

We report a modified digestion method that improves the recovery of Toxocara canis larvae from skeletal muscle. Minced muscle tissue from infected mice was incubated in artificial gastric juice for 48 hr at 37 C, and ethanol was added for the second 24 hr. This procedure allowed the larvae to be identified and counted more quickly than with the standard digestion method. This method allows measurement of the total number of larvae present in muscle tissue following oral intubation of embryonated eggs, although it does not permit counting of live larvae.

Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology.

Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in DeltaScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of DeltaScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in DeltaScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

Prevention of interstitial fibrosis of renal allograft by angiotensin II blockade.

We previously confirmed that losartan (LOS), an angiotensin-II (A-II) receptor blocker, diminished plasminogen activator inhibitor-1 (PAI-1) in cyclosporine (CsA)-treated renal graft recipients. Because PAI-1 is known to correlate with tissue fibrosis, we speculated that LOS would have the potential to prevent renal graft interstitial fibrosis. In this study, we focused our attention on the LOS-induced histopathologic changes in renal grafts. Out of 24 CsA-treated normotensive kidney transplanted patients, 8 began to take 25 to 50 mg/day of LOS soon after kidney transplantation (group 1). Eight did so 2 years after kidney transplantation (group 2). Eight received no ARBs as a control group (group 3). PAI-1 levels were monitored every 3 months for 2 years. Renal graft biopsy was performed on all participants, with informed consent, before and 2 years after the onset of this study. The biopsy specimens were stained with periodic acid-methenamine-silver (PAM)-Masson stain for light-microscopic examination. Fibrotic areas in each biopsy specimen were measured using the LUZEX-III image analyzing system. Statistical analysis was performed using Student's t-test. When we considered the pre-value of PAI-1 in each patient as 100%, the mean percent value of PAI-1 at 2 years after the onset of this study of groups 1, 2, and 3 were 81.5 +/- 10.3%, 90.1 +/- 12.5%, and 116.8 +/- 11.9%, respectively (P < .01 groups 1 and 2 vs group 3). Light-microscopic examination revealed less remarkable renal interstitial fibrosis among LOS administered groups. A-II blockade may be a key to prevent renal graft interstitial fibrosis.

A microbeam X-ray diffraction study of insulin spherulites.

The peptide hormone insulin forms a spherical aggregate, called a spherulite, at low pH and high temperature. A spherulite is composed of a core and many fibrils extending from it. These fibrils are thought to be amyloid fibers with a beta-sheet structure. In the present study, spherulites with a diameter of 50-100 microm were examined by X-ray fiber diffraction using a 6 microm beam. When a spherulite was scanned with the microbeam and the observed diffraction patterns were arranged in a two-dimensional array, the direction of the scatter was centrosymmetric, demonstrating a symmetric growth of fibrils. There were diffraction peaks at Bragg spacings of 23 nm, 3.3 nm and 1.2 nm in the direction perpendicular to the fibrils and 0.48 nm along the fibrils. The 0.48 nm reflection shows that the hydrogen bonds between beta-strands are along the fibril. The 23 nm reflection corresponds to the separation between fibrils, the 3.3 nm reflection is due to the arrangement of protofilaments, and the 1.2 nm reflection arises from the arrangement of peptide chains. On the basis of these results, a model of a fibril with an extended insulin molecule is proposed.

Expression of glucocorticoid receptors in non-neoplastic lymphoid follicles and B cell type malignant lymphomas.

OBJECTIVE: To evaluate the expression of human glucocorticoid receptors (hGRs), such as hGR (4H2), hGR-alpha, and hGR-beta, in non-neoplastic lymphoid follicles and B cell type malignant lymphomas. METHODS: The expression of hGRs in non-neoplastic lymphoid follicles and malignant lymphomas, including diffuse large cell lymphoma, mantle cell lymphoma, and follicular lymphoma, was examined immunohistochemically. HGR (4H2) expression was confirmed by double immunostaining of tissues and in isolated cells from tonsillar germinal centres, and by immunoelectronmicroscopy. RESULTS: In secondary lymphoid follicles of any non-neoplastic diseases--such as chronic tonsillitis, reactive lymphadenitis, and Kimura's disease--the germinal centre cells often expressed hGR (4H2) and hGR-alpha. Double immunocytochemical staining of isolated germinal centre cells showed that the majority of hGR (4H2) positive cells were CD20 positive B cells, and that follicular dendritic cells also expressed hGR. Immunoelectronmicroscopy revealed the presence of nuclear hGR (4H2) in the binucleated follicular dendritic cells and germinal centre cells. The frequency of hGR (4H2) expression in diffuse large B cell lymphoma was higher, that in mantle cell lymphoma was lower, and that in follicular lymphoma was intermediate among the types of malignant lymphoma. The hGR (4H2) expression was less frequent in cases of grade I follicular lymphoma. CONCLUSIONS: There are differences in hGR expression between the germinal centre and the mantle zone in non-neoplastic lymphoid follicles, and differences of hGR (4H2) expression among the types of malignant lymphoma and grades of follicular lymphoma, which probably contribute to the different steroid sensitivities.

Successive changes in tissue migration capacity of developing larvae of an intestinal nematode, Strongyloides venezuelensis.

Infective larvae of an intestinal nematode, Strongyloides venezuelensis, enter rodent hosts percutaneously, and migrate through connective tissues and lungs. Then they arrive at the small intestine, where they reach maturity. It is not known how S. venezuelensis larvae develop during tissue migration. Here we demonstrate that tissue invasion ability of S. venezuelensis larvae changes drastically during tissue migration, and that the changes are associated with stage-specific protein expression. Infective larvae, connective tissue larvae, lung larvae, and mucosal larvae were used to infect mice by various infection methods, including percutaneous, subcutaneous, oral, and intraduodenal inoculation. Among different migration stages, only infective larvae penetrated mouse skin. Larvae, once inside the host, quickly lost skin penetration ability, which was associated with the disappearance of an infective larva-specific metalloprotease. Migrating larvae had connective tissue migration ability until in the lungs, where larvae became able to settle down in the intestinal mucosa. Lung larvae and mucosal larvae were capable of producing and secreting adhesion molecules.

[Norwood procedure after colostomy; report of a case].

We encountered a baby who was diagnosed with anal atresia, interruption of aortic arch (type B), aortic stenosis, mitral stenosis, single atrium, large ventricular septal defects, aberrant origin right subclavian artery. We operated on him using the Norwood procedure after a colostomy. Aortic arch was reconstructed by interposing with an 8 mm graft and the right ventricular-pulmonary artery (RV-PA) conduit was chosen for pulmonary flow. We closed the sternum 6 days after the Norwood procedure. We extubated him 16 days after delayed sternal closure. There was no trouble with his stoma and no sign of infection. The postoperative echocardiography didn't show the finding which left pulmonary artery was stenotic, but the lung perfusion schintigraphy revealed an imbalance in the distribution of lung perfusion. He was discharged 70 days after undergoing the Norwood procedure.

Inhibition of plasminogen activator inhibitor-1 by angiotensin II receptor blockers on cyclosporine-treated renal allograft recipients.

INTRODUCTION: We previously showed that proteinuria from a renal graft was significantly decreased by administration of losartan potassium, an angiotensin II receptor blockers (ARB). To further evaluate the mechanism, we performed another clinical study focusing on the change in plasma plasminogen activator inhibitor-1 (PAI-1) levels among cyclosporine (CyA)-treated renal allograft recipients. METHODS: Among 12 hypertensive CyA-treated kidney transplant patients, four received 25 to 50 mg/day of losartan; four, 4 to 8 mg/day of candesartan cilexetil; and another four, 20 to 40 mg/day of nifedipine. Four CyA-treated kidney-transplanted patients without hypertension were selected as a control group. Informed consent was obtained from all participants. PAI-1 and serum creatinine (S-Cr) levels were monitored every 3 months for 1 year. RESULTS: Considering the pretreatment of PAI-1 as 100%, the mean percent of PAI-1 at 1 year after the onset of study for losartan, candesartan, nifedipine, and control groups were 78.6 +/- 6.7%, 81.4 +/- 8.0%, 96.7 +/- 7.6%, and 110.4 +/- 9.2%, respectively. The ARB groups demonstrated significant differences from the control group (P < .01), while the nifedipine group did not. S-Cr levels among ARB-administered groups were increased slightly but temporarily. As for S-Cr levels, no significant differences were seen among the four groups. CONCLUSIONS: Control of hypertension itself is important for all renal graft recipients; however, PAI-1 reduction by ARBs was thought to be a key for renal preservation. We expect that ARBs will contribute to prolonged renal allograft survival.

Infant schistosomiasis in Ghana: a survey in an irrigation community.

We used a rapid, visually read, field applicable monoclonal antibody (MoAb)-dipstick assay for specific diagnosis of urinary schistosomiasis together with microscopy to determine the prevalence of infant schistosomiasis in a community in the Awutu-Efutu Senya District in the Central Region of Ghana. The study group consisted of 97 infants (51 males and 46 females) aged 2 months to 5 years. A total of 75 of 97 (77.3%) subjects submitted stool samples; none had Schistosoma mansoni. Three individuals (3.1%) had hookworms but there were no other intestinal helminths. The urinary schistosomiasis prevalence by MoAb-dipstick (30%) was higher (P < 0.05) than that estimated by microscopy (11.2%). However, three of nine (33.3%) microscopically confirmed cases tested MoAb-dipstick positive after pre-treatment of the urine specimen with heat. The youngest infant to be found infected with S. haematobium microscopically was 4 months old. Fifteen of 71 S. haematobium egg negative individuals tested dipstick positive, giving a dipstick specificity of 78.9% as compared with microscopy as gold standard test. The relative sensitivity of the dipstick was 100%.

[New surgical approach "intrapulmonary septation technic" for Fontan candidates with unilateral pulmonary arterial hypoplasia and/or pulmonary venous obstruction].

Unilateral pulmonary arterial hypoplasia and/or pulmonary venous obstruction are serious hazards for Fontan candidates. For these patients, we have started new surgical approach "intrapulmonary septation technic". This consists of 3 components; (A) partial right heart bypass to well-grown side, (B) mandatory pulmonary blood flow to low-capacity side, (aorto-pulmonary shunt or others) and (C) a patch between partial right heart bypass and mandatory pulmonary blood flow. Thirteen patients underwent the approach. The source of partial right heart bypass was brought from superior vena cava (11 patients), inferior vena cava (1 patient) and fenestrated Fontan (1 patient). The mandatory pulmonary blood flow was supplied by aorto-pulmonary shunt (11 patients), pulmonary arterial banding (1 patient) and native pulmonary valve stenosis (1 patient). We added pulmonary artery enlargement (9 patients), release of pulmonary venous obstruction (8 patients) and/or atrio-ventricular valve plasty (5 patients), simultaneously. No hospital death. Early post-operative course was uneventful in all cases except 1, as pulmonary blood flow to low-capacity side had increased gradually after this intervention. Eight patients had reached Fontan operation. In this approach, nearly whole pulmonary artery can grow without any affect of volume overload through well-grown side from collateral arteries of low-capacity side. All procedures of "intrapulmonary septation technic" and reconstruction of pulmonary artery in Fontan operation can be easily performed in larger pulmonary artery of well-grown side, eliminating need for extensive dissection.