Multistate outbreak of Listeria monocytogenes infections linked to whole apples used in commercially produced, prepackaged caramel apples: United States, 2014-2015.

Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. We investigated a nationwide listeriosis outbreak associated with caramel apples. We defined an outbreak-associated case as an infection with one or both of two outbreak strains of Listeria monocytogenes highly related by whole-genome multilocus sequence typing (wgMLST) from 1 October 2014 to 1 February 2015. Single-interviewer open-ended interviews identified the source. Outbreak-associated cases were compared with non-outbreak-associated cases and traceback and environmental investigations were performed. We identified 35 outbreak-associated cases in 12 states; 34 (97%) were hospitalized and seven (20%) died. Outbreak-associated ill persons were more likely to have eaten commercially produced, prepackaged caramel apples (odds ratio 326·7, 95% confidence interval 32·2-3314). Environmental samples from the grower's packing facility and distribution-chain whole apples yielded isolates highly related to outbreak isolates by wgMLST. This outbreak highlights the importance of minimizing produce contamination with L. monocytogenes. Investigators should perform single-interviewer open-ended interviews when a food is not readily identified.

Multistate outbreak of listeriosis caused by imported cheese and evidence of cross-contamination of other cheeses, USA, 2012.

Listeria monocytogenes is a foodborne pathogen that can cause bacteraemia, meningitis, and complications during pregnancy. In July 2012, molecular subtyping identified indistinguishable L. monocytogenes isolates from six patients and two samples of different cut and repackaged cheeses. A multistate outbreak investigation was initiated. Initial analyses identified an association between eating soft cheese and outbreak-related illness (odds ratio 17·3, 95% confidence interval 2·0-825·7) but no common brand. Cheese inventory data from locations where patients bought cheese and an additional location where repackaged cheese yielded the outbreak strain were compared to identify cheeses for microbiological sampling. Intact packages of imported ricotta salata yielded the outbreak strain. Fourteen jurisdictions reported 22 cases from March-October 2012, including four deaths and a fetal loss. Six patients ultimately reported eating ricotta salata; another reported eating cheese likely cut with equipment also used for contaminated ricotta salata, and nine more reported eating other cheeses that might also have been cross-contaminated. An FDA import alert and US and international recalls followed. Epidemiology-directed microbiological testing of suspect cheeses helped identify the outbreak source. Cross-contamination of cheese highlights the importance of using validated disinfectant protocols and routine cleaning and sanitizing after cutting each block or wheel.

Microsatellite markers for Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) and other Cryptolestes species.

Cryptolestes ferrugineus (Stephens, 1831) is an important insect pest of stored products. Due to its broad host range, short life cycle, and high reproductive capacity, this species has rapidly colonized temperate and tropical regions around the world. In this study, we isolated 18 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. These loci will be useful tool to better understand the genetic structure and migration patterns of C. ferrugineus throughout the world. The genetic parameters were estimated based on 80 individual C. ferrugineus from two natural populations. The results revealed that 18 loci were different polymorphic levels. The numbers of alleles ranged from 3 to 12, and eleven loci demonstrated polymorphic information contents greater than 0.5. The observed (H O) and expected (H E) heterozygosities ranged from 0.051 to 0.883 and 0.173 to 0.815, respectively. Five locus/population combinations significantly deviated from Hardy-Weinberg equilibrium. We also demonstrated the potential utility of the C. ferrugineus microsatellites as population and species markers for four additional Cryptolestes species.

COI barcode based species-specific primers for identification of five species of stored-product pests from genus Cryptolestes (Coleoptera: Laemophloeidae).

Flat grain beetles of the genus Cryptolestes (Coleoptera: Laemophloeidae) are one of the economically most important stored-product pests which feed on many kinds of agricultural products, especially grains. Nine of more than 40 described Cryptolestes species are recognized as stored-product pests and two of the pest species have a cosmopolitan distribution. Given the rapid growth in global trade of food products, ecological barriers to the spread of pests are easily overcome. Therefore, development of reliable systems for routine quarantine inspection and early infestation detection is vital. In the present study, we established a new rapid and accurate cytochrome c oxidase subunit I-based system for molecular identification of five common stored-product Cryptolestes species, namely, Cryptolestes capensis, Cryptolestes ferrugineus, Cryptolestes pusilloides, Cryptolestes pusillus and Cryptolestes turcicus. Five species-specific primer pairs for traditional uniplex polymerase chain reaction assay are described and their specificity and sensitivity for the identification process is evaluated using larval samples of 12 different populations from three continents (Asia, Europe and North America).

DNA barcoding of five common stored-product pest species of genus Cryptolestes (Coleoptera: Laemophloeidae).

Several species of the genus Cryptolestes Ganglbauer, 1899 (Coleoptera: Laemophloeidae) are commonly found in stored products. In this study, five species of Cryptolestes, with almost worldwide distribution, were obtained from laboratories in China, Czech Republic and the USA: Cryptolestes ferrugineus (Stephens, 1831), Cryptolestes pusillus (Schönherr, 1817), Cryptolestes turcicus (Grouvelle, 1876), Cryptolestes pusilloides (Steel & Howe, 1952) and Cryptolestes capensis (Waltl, 1834). Molecular identification based on a 658 bp fragment from the mitochondrial DNA cytochrome c oxidase subunit I (COI) was adopted to overcome some problems of morphological identification of Cryptolestes species. The utility of COI sequences as DNA barcodes in discriminating the five Cryptolestes species was evaluated on adults and larvae by analysing Kimura 2-parameter distances, phylogenetic tree and haplotype networks. The results showed that molecular approaches based on DNA barcodes were able to accurately identify these species. This is the first study using DNA barcoding to identify Cryptolestes species and the gathered DNA sequences will complement the biological barcode database.

Size exclusion chromatography as a tool for evaluation of fragmentation pattern of gastric mucins under non-degrading conditions.

Gastric mucins are high molecular weight extracellular glycoproteins that play a major role in the protection of the gastrointestinal tract and besides that they are also involved in many disease processes. In the present study, size exclusion chromatography under non-degrading conditions was used to study the fragmentation pattern of native gastric mucins. The samples of gastric mucins of different origin obtained by an extraction of gastric mucosa with Tris-HCl buffer, pH 7.3 were separated using size exclusion chromatography on Sephadex G-100. While samples of rat gastric mucins are characterized by the presence of only high-molecular weight fraction of glycoproteins, fragmented mucin components in non-denaturated samples were observed in canine and human gastric mucins. Differences in the fragmentation pattern were observed in patients with ulcer diseases and gastric cancer. Degradation products of mucins were also detected using polyacrylamide gel electrophoresis in the presence of SDS.

Mechanical and microwave absorbing properties of carbon-filled polyurethane.

Polyurethane (PU) matrix composites were prepared with various carbon fillers at different filler contents in order to investigate their structure, mechanical and microwave absorbing properties. As fillers, flat carbon microparticles, carbon microfibers and multiwalled carbon nanotubes (MWNT) were used. The microstructure of the composite was examined by scanning electron microscopy and transmission electron microscopy. Mechanical properties, namely universal hardness, plastic hardness, elastic modulus and creep were assessed by means of depth sensing indentation test. Mechanical properties of PU composite filled with different fillers were investigated and the composite always exhibited higher hardness, elastic modulus and creep resistance than un-filled PU. Influence of filler shape, content and dispersion was also investigated.

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine.

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases.

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.

Immobilized-metal-ion affinity chromatography as a tool for the qualitative study of pepsinogen phosphorylation.

Affinity chromatography on immobilized Fe(3+) ions--immobilized-metal-ion affinity chromatography (IMAC) method--was used for the determination of pepsin and pepsinogen phosphorylation. IMAC is a very powerful method for detailed studies of proteins. Dephosphorylation of the pepsinogens and pepsins has no effect on their proteolytic ability. For this reason, the determination of proteolytic activity was used for the detection of pepsinogen (pepsin) presence in the collected fractions as a very suitable and specific method. Pepsins and their zymogens probably have the same amounts of phosphate ions in their molecule. The exact definition of conditions is very important for the prepurification of the proteinases and for their analysis.

Fat and vitamin A stability in the presence of Ronoxan A and other antioxidants.

The effect of the antioxidant Ronoxan A and of two of its components (ascorbyl palmitate, lecithin) and of tocopheryl acetate was tested for the stabilization of fats and retinyl acetate. In the present study, results obtained in sunflower oil, soybean oil, milk fat and in the mixtures of these fats in concentrations corresponding to their levels in the Czechoslovak Infant Formula (Feminar) are compiled. The antioxidant levels applied, i.e. those used in Czechoslovakia for the stabilization and enrichment of vegetable oils and Feminar, have a protective effect on retinyl acetate to a certain extent as well, and in no case they affect it unfavourably. All antioxidant combinations applied cause a significant retardation of the fat autoxidation. Ronoxan A has the best effect in all cases tested.

Antigenic characterization of human isolates of cryptosporidia.

Isolates of cryptosporidia from immunodeficient patients and from immunocompetent children suffering from diarrhoea were sources for preparation of antigens. Altogether, antigens from 21 isolates were obtained, 19 from children and 2 from AIDS patients. With one exception, all children were under 4 years old and most of them were between 1 and 2 years old. The probable source of infection was found in 11 cases. In 8 cases, as well as in both AIDS patients, the source of infection was not determined. The same groups of persons and the control group of healthy blood donors were examined serologically using ELISA with the antigen of bovine origin and positive sera were used for following experiments. Soluble and membrane-enriched antigens from oocysts were compared using polyacrylamide gel electrophoresis and electroimmunotransfer blots developed by human immune sera. While no differences were recorded in soluble antigens, two of the membrane-enriched antigens were missing (15.5 and 33 kD bands). While all isolates with a pattern typical for Cryptosporidium parvum were infective for neonatal mice, both isolates with missing bands were not infective for newborn mice in repeated experiments. The first of them was infective for chickens and was originally determined to be Cryptosporidium baileyi (Ditrich et al. 1991). However, the antigenic pattern differs from pattern of this species. The second isolate was infective for guinea pigs and its species classification remains uncertain.

The severe combined immunodeficient mouse as a model for Encephalitozoon cuniculi microsporidiosis.

Microsporidia have been recently recognized as opportunistic pathogens in AIDS patients. In attempt to develop an animal model with features similar to the infections observed in the immunodeficient patients, the adult severe combined immunodeficient mice (SCID) were administered both intraperitoneally and perorally by 2 x 10(7) spores of the murine isolate of E. cuniculi. The experimental inoculation caused a severe, fatal disease characterized by the dissemination of microsporidia into the host tissues. The dominant route of E. cuniculi dissemination in the SCID mice was continual direct extension from the site of inoculation to adjacent tissues and organs, terminating in hematogenous spread of infection in the host. The different courses of microsporidiosis in SCID mice relative to the mode of inoculation (i.p. vs. p.o.) was observed. The survival time of i.p. infected SCID mice was 3 weeks--vs. 5 weeks in p.o. infected SCID mice. Experimental microsporidiosis in SCID mice should provide a useful model for studies in microsporidial pathogenesis, mechanisms of resistance, immunotherapy, and in evaluating potential antimicrosporidial agents.

Prophylactic and therapeutic immune reconstitution of SCID mice infected with Encephalitozoon cuniculi.

Severe combined immunodeficient (SCID) mice develop lethal infections, resembling opportunistic microsporidiosis of immunocompromised patients, after intraperitoneal (i.p.) inoculations of spores of Encephalitozoon cuniculi. In the present study, SCID mice reconstituted i.p. with 5 x 10(7) spleen cells from naive adult BALB/c mice 14 days prior to the i.p. injection of 10(7) spores were completely resistant to the infection, whereas control infected SCID mice developed clinical disease and died within 17 days post infection (DPI). In another experiment, SCID mice infected i.p. with 10(7) spores of E. cuniculi and after that (on DPI 7) injected i.p. with 5 x 10(7) spleen lymphocytes isolated from immune adult BALB/c mice were partially protected against the parasite (40% of the reconstituted mice survived). In both experiments, high levels of parasite-specific serum antibodies (mostly of the IgG-isotype) were detected in the infected immunocompetent BALB/c mice, whereas virtually no antibodies were found in the infected SCID mice. However, SCID mice reconstituted with either naive spleen cells or immune lymphocytes revealed humoral immune responses comparable with those of immunocompetent mice.

[Immunohistologic detection of pepsin and gastricsin in carcinoma of the stomach]. / Imunohistologický prukaz pepsinu a gastricsinu v karcinomech zaludku.

Immunohistological tests for pepsin and gastricsin were carried out in bioptic samples of 51 patients with carcinoma of the stomach. Pepsin was detected in only 2 cases (4%), gastricsin in 26 patients (55%). Compared to the histological picture, the rate of incidence of gastricsin showed no difference between the intestinal and diffuse types of this tumour. In all examined cases, only gelatinous carcinomas were negative. Nor were any differences found in the cardia, the body or antrum. Surprisingly, these enzymes were found in the cytoplasma of neutrophylic granulocytes in the inflammatory infiltrate of the mucosal and tumorous stroma. Intestinal metaplasia of the epithelium was always negative even in the neighbourhood of positive tumours. The detected changes are evidence against the possibility of different histogenesis of the diffuse and intestinal forms of carcinoma of the stomach as well as against the possibility that intestinal metaplasia in a chronic inlammation of the stomach could be regarded as a direct first stage of the intestinal form of carcinoma of the stomach. In cases of metastases of unknown origin, pepsin and gastricsin cannot serve as markers due to their insufficient organ and cell specificities.

[Pepsinogen C in the human prostate]. / Pepsinogen C v lidské prostate.

The authors investigated the proteolytic activity of the normal prostate, the prostate with benign hypertrophy and with cancer. Pepsinogen obtained from the normal prostate was isolated on DEAHP pearl cellulose and purified by means of affinite chromatography. Separation on affinite chromatography and by electrophoresis on agar confirmed that the pepsinogen is identical with gastric progastricsin. The proteolytic activity was examined by a modified method of Anson and Mirsky, and with the aid of synthetic substrates it was assessed quantitatively in all three examined groups. The enzyme activity of the carcinomatous tissue practically did not differ from the activity of the normal prostate. In beniming hypertrophy of the prostate the proteolytic activity assessed by the breakdown of haemoglobin is markedly reduced and when tested on the breakdown of synthetic substrates [For-Tyr-Tyr- and Ac-Phe- Tyr (I2)] it is practically zero.

Resistance to selected beta-lactam antibiotics.

Susceptibility in vitro and trends in resistance to antimicrobials were determined by a dilution micromethod in a group of Actinobacillus pleuropneumoniae, Pasteurella multocida, Mannheimia haemolytica and Escherichia coli isolates from clinical cases of cattle and swine diseases in the Czech Republic from 2007 to 2011. A high susceptibility of pig and cattle respiratory pathogens to antimicrobials was found, with the exception of the moderate prevalence of M. haemolytica resistance to ampicillin. In contrast to respiratory pathogens, low susceptibility of E. coli of pig and cattle isolates to ampicillin and amoxicillin/clavulanic acid was noted. Regarding resistance trends, an increase in levels of resistance among E. coli isolates to ampicillin and amoxicillin/clavulanic acid was identified, but the resistance of respiratory isolates was low, with the exception of M. haemolytica. For the period of 2007-2011, there was a significant and almost continuous increase in sales (compared with population correction unit) of ceftiofur, cefquinome and other beta lactams for pigs. Consumption peaked in 2010. In the case of amoxicillin in combination with clavulanic acid, data showed a significant decrease in sales from 2007 to 2008, followed by a period of fluctuation. In cattle, within the groups of 3rd and 4th generation cephalosporins and for the whole group of other betalactams for the period of 2007-2011, there was a significant and almost continuous increase in sales (compared with population correction unit). Consumption peaked in 2010. In the case of ceftiofur, there was a huge increase noted from 2010. In the case of amoxicillin in combination with betalactamase inhibitor (clavulanic acid) data shows a significant decrease from 2007 to 2008, followed by a period of fluctuation in sales.

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolates from clinical outbreaks of porcine respiratory diseases.

Limited data regarding the susceptibility of Actinobacillus pleuropneumoniae to antimicrobials has been published during recent years. Accordingly, the aim of the present study was to investigate the distribution of MICs for the isolates of A. pleuropneumoniae from diseased pigs in the Czech Republic between 2007 and 2009. A total of 242 isolates were tested for susceptibility to 16 antimicrobial agents by a broth microdilution method. A low degree of resistance was observed for florfenicol (0.8%), amoxicillin and clavulanic acid (0.8%), tilmicosin (1.2%), tiamulin (1.7%) and ampicillin (3.3%), whereas resistance to tetracycline was detected more frequently, 23.9% of isolates. Interestingly, resistance to florfenicol has not yet been reported in any study investigating antimicrobial resistance of A. pleuropneumoniae. By PCR the presence of the floR gene was confirmed in all florfenicol resistant isolates.

Passive immunisation of post-weaned piglets using hyperimmune serum against experimental Haemophilus parasuis infection.

The protective role of hyperimmune serum in the prevention of Haemophilus parasuis infections in post-weaned piglets was assessed by experimental challenge. The hyperimmune serum was obtained from a pig vaccinated with a commercial vaccine against Glässer's disease. Thirty-eight weaned piglets were divided into four groups: three groups were immunised intramuscularly with 10 ml of hyperimmune serum and one group consisted of unimmunised control animals. All piglets were subsequently infected intraperitoneally with H. parasuis serotype 5 at different times after immunisation. The use of hyperimmune serum provided the piglets with partial protection against experimental infection. The levels of protection indirectly depend on time between serum inoculation and challenge infection. The best protection of piglets against experimental infection was obtained in the group immunised 1 week before inoculation; the same group in which the highest levels of antibodies were detected at the time of challenge.

Microsporidiosis and Cryptosporidiosis in HIV/AIDS Patients in St. Petersburg, Russia: Serological identification of microsporidia and Cryptosporidium parvum in sera samples from HIV/AIDS patients.

To determine seroprevalence of the opportunistic organisms Cryptosporidium parvum and microsporidia (Encephalitozoon cuniculi, E. intestinalis, E. hellem, and Enterocytozoon bieneusi) in Russian HIV/AIDS patients, we evaluated 46 sera from HIV/AIDS patients from the S.P. Botkin Clinical Infectious Diseases Hospital, St. Petersburg, Russia. Five (10.9%) sera were seropositive for E. cuniculi and 19 (41.3%) were positive for C. parvum by ELISA. By IFAT, 6 (13.0%) sera were seropositive for E. bieneusi, 4 (8.7%) for E. intestinalis, and 9 (19.6%) for E. hellem. This study is the first report to estimate the prevalence of infection with Cryptosporidium and microsporidia among Russian HIV/AIDS patients.