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.

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.

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.

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.

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.

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.

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.

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.

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.

Comparison of different doses of antigen for intradermal administration in pigs: the Actinobacillus pleuropneumoniae model.

The intensity of antibody responses and level of protection against challenge infection induced by Actinobacillus pleuropneumoniae (APP) were compared in piglets vaccinated intramuscularly with different vaccination doses of a subunit vaccine. Secondary antibody responses to APP exotoxins induced by the intradermally administered diluted vaccines did not differ from those induced by undiluted vaccines administered either intradermally or intramuscularly. The level of protection measured by the clinical course of challenge infection to the extent of lung lesions was significantly higher in animals vaccinated with a three-time diluted vaccine in comparison with animals that were administered intramuscularly or intradermally with the most concentrated or the most diluted vaccines.

Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity.

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.

Identification of Pasteurella multocida Serogroup F isolates in rabbits.

A total of 24 Pasteurella multocida rabbit isolates obtained from 24 rabbit flocks in the Czech Republic during the period of between 2001 and 2004 were analysed by capsular PCR typing. Apart from isolates identified as serogroups A (n = 14, 58.4%) and D (n = 2, 8.3%), eight isolates (33.3%) were identified as members of serogroup F. This serogroup had been predominantly associated with poultry infections so far. The rabbit serogroup F isolates were characterized in detail by ribotyping with restriction to endonuclease MspI revealing two distinct ribotypes. Seven serogroup F isolates were assigned to ribotype 1 and one isolate was assigned to ribotype 2.

Identification of surface-membrane P-type ATPases resembling fungal K(+)- and Na(+)-ATPases, in Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani.

Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-, proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na(+) or K(+) ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na(+)-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei.

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.

Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized alpha-chymotrypsin.

Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was used for the comparison of peptide maps of pepsin after its digestions by different forms of immobilized alpha-chymotrypsin. Porcine pepsin was hydrolysed with soluble alpha-chymotrypsin, with alpha-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with alpha-chymotrypsin attached to poly(acrylamide-allyl glycoside) copolymer or to glycosylated hydroxyalkyl methacrylate copolymer Separon or to agarose gel Sepharose 4B. Efficiency of enzymatic protein cleavage with regard to peptide mapping of porcine pepsin has been examined by the use of alpha-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with alpha-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. Alpha-chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified alpha-chymotrypsin was not suitable for efficient pepsin cleavage.

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.

Study of human pepsinogen polymorphism by combining chromatographic and electrophoretic methods.

A new combination of chromatographic and electrophoretic methods has been developed for better separation and characterization of human pepsinogens. Pepsinogens isolated from the gastric mucosa of patients with gastric cancer have been separated using fast-protein liquid chromatography (FPLC) on an ionex Uno-Q1 column. Proteolytic active fractions were firstly immunodetected by monoclonal antibodies against PGA and PGC using ELISA and then separated by isoelectric focusation in the acidic pH 2.5-5 gradient with an excellent resolution. The combination FPLC and ELISA followed by IEF enabled to separate ten pepsinogen isoforms. This technique is very suitable for studies of the pepsinogen polymorphism and its role in the gastric diseases.