Unique response characteristics in persistent strains of Listeria monocytogenes exposed to low pH.

Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.

The concentration of iodine in horse serum and its relationship with thyroxin concentration by geological difference.

In this study, iodine and thyroxin (T4) concentrations in the serum of 69 horses were investigated. Higher iodine concentrations were obtained from the horses housed in Chiba Prefecture. In contrast, T4 concentrations of horses at Shizuoka Prefecture were higher than those of horses at Chiba Prefecture. There was a significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations of horses at Saitama and Shizuoka prefectures. Although a significant correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefecture, the distribution area of the data was separated from the data of horses housed in Saitama and Shizuoka prefectures. A higher iodine concentration in the environment is expected in the sampling area at Chiba Prefecture. Thus, it was suggested that the concentrations of iodine in the serum of horses are influenced by geological differences. It was thought that equine serum is a useful sample for monitoring.

Investigation of cadmium contamination using hair of the Japanese macaque, Macaca fuscata, from Shimokita Peninsula, Aomori Prefecture in Japan.

The cadmium (Cd) contents in hair of macaques (n = 45, Macaca fuscata) living on the Shimokita Peninsula were investigated. The mean Cd contents in the hair of Japanese (n = 34, 5.01 µg/g) and macaques (3.05 µg/g) tendency to be higher than those of animals living other areas. The Cd contents of hair of wild macaques were significantly (p < 0.01) lower than that of humans, although three were no significant difference between Cd contents of humans and that of the macaque in captivity. The hair of the macaque was suggested as a useful sample for measurement of Cd contamination in the environment.

Prevalence of Listeria monocytogenes in retailed meat in the Tokyo metropolitan area.

This study was conducted to determine the prevalence of Listeria monocytogenes in retailed meats, comprising beef, chicken, and pork, in the Tokyo metropolitan area. A total of 379 samples of retailed meat were collected from 1998 to 2003, most of which were obtained by simultaneously purchasing the three classes of meat from a shop and then making another simultaneous purchase of meat from the same shop a few weeks later. The prevalence of L. monocytogenes was 28.0%, and the serotypes isolated were mainly 1/2a, 1/2b, 1/2c, and 4b. Comparison of the prevalence of each serotype among the classes of meat showed a predominant distribution of serotypes 1/2a, 1/2b, and 4b in chicken, while serotype 1/2c was dominant in pork. A total of nine cases considered to be due to persistence and/or cross-contamination were found. Most of the strains involved in persistence and/or cross-contamination were of serotypes 1/2c or 4b. These results suggest that contamination in retailed meat in Japan is at almost the same level as in other countries and that chicken has the highest potential as a source of contamination and infection. In addition, we suggest that the ecological niche of serotype 1/2c is distinct from those of 1/2a, 1/2b, and 4b, which may explain why human hosts have less opportunity to be exposed to serotype 1/2c and why there is a lower rate of isolation of this serotype from cases of human listeriosis.

Genetic variation of mitochondrial DNA in Phalacrocorax carbo in Japan.

A Japanese resident bird, Phalacrocorax carbo hanedae (Japanese name: Kawa-u), was threatened with extinction due to deterioration of its habitat in the 1970s, but the population has since recovered thanks to environmental protection measures. This study analyzed the genetic diversity of 18 Kawa-u individuals living in the basins of the Abe and Warashina rivers in Shizuoka Prefecture, Japan. We obtained seven haplotypes of mitochondrial D-loop sequences and compared them with 49 European P. carbo D-loop haplotypes. We identified four new haplotypes but no clear genetic evidence distinguishing the Kawa-u as a distinct subspecies of P. carbo. Our results suggest the need for further surveillance of the P. carbo genetic lineage, regardless of the geographical distribution.

Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera.

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan.

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.

Sequence-Based Characterization of Listeria monocytogenes Strains Isolated from Domestic Retail Meat in the Tokyo Metropolitan Area of Japan.

The level of Listeria monocytogenes contamination of domestic retail meat in Tokyo, Japan, was assessed by comparison of isolates from 2004 to 2007 with those isolated before 2003. The overall prevalence of L. monocytogenes among these samples significantly diminished over time (1998-2003, 28.0%; 2004-2007, 17.6%) reflecting a significant decrease in the frequency of contamination of beef. Serotype 1/2a was isolated most frequently, reflecting a change in the predominant serotype in pork from 1/2c to 1/2a. We performed a simple genetic subtyping method based on 3 genes, iap, sigB, and actA, as well as traditional multilocus sequence typing to classify the allele types (ATs). No extensive variation among sequence types was detected. However, increased genetic diversity among the ATs of the 3 genes in the 2004-2007 isolates was evident. We identified AT 26 of the iap gene, which was not previously reported in Japanese isolates, and 6 ATs of the sigB gene, including 4 with nonsense mutations not currently registered in L. monocytogenes DNA databases. sigB is an evolutionally conserved gene that plays a role in the stress response. Our results indicate that the sigB gene may be relatively unstable among L. monocytogenes strains circulating in Japan.

Isolation of Listeria monocytogenes from the skin of slaughtered beef cattle.

We attempted to isolate Listeria monocytogenes from skin, contents of large intestines and carcasses of cattle introduced to a slaughterhouse in order to identify source of contamination for this pathogen. Sixty skin samples, 60 samples of the contents of large intestines and 30 carcass samples were colleted in June, August and November 2003 for use in this study. Listeria spp. and L. monocytogenes were isolated from 30 (50%) and 3 (5%) of the cattle skin samples, respectively. However, no Listeria spp., including L. monocytogenes, were isolated from intestinal contents or carcasses. Seven isolates were obtained, of which five and two strains were serotypes 1/2a and 1/2b, respectively. Genetic analysis suggested that there was persistent inhabitation of the pathogen around the area investigated in this study.

Genetic subtyping of Listeria monocytogenes via multiple-locus sequence typing using iap, sigB and actA.

Pulse field gel electrophoresis (PFGE) is widely used for listeriosis surveillance. Although this technique is effective for epidemiology, the data among laboratories are inconsistent. We previously reported a method for Listeria monocytogenes subtyping combined with sequence analysis of partial iap and whole genome restriction fragment length polymorphism (RFLP) using XbaI, ClaI (BanIII) and PstI. However, distinguishing subtypes was challenging, because the output comprised complicated fragment patterns. In this study, we aimed to establish a simple genotyping method that does not depend on visual observation, rather it focuses on multi-locus sequence typing (MLST) using three genes, iap, sigB and actA. Sixty-eight strains of L. monocytogenes including EGD-e as a reference strain were investigated to ensure consistency with previous data on the genetic characterization. All strains were grouped into 29 types by both analyses. Although there are some differences in classification, major clades included the same strains. Simpson's indices of diversity (SID) by MLST and iap-RFLP-based typing were 0.967 (95% confidence interval [CI]: 0.955/0.978) and 0.967 (95% CI: 0.955/0.979), respectively. The discriminatory power of both methods can be considered almost identical. Compared with the results of 38 selected strains, the strains within the MLST clusters in this study coincided with those obtained using PFGE. Thus, the MLST strategy could help differentiate among L. monocytogenes isolates during epidemiological studies.

Tissue toxicokinetics of perfluoro compounds with single and chronic low doses in male rats.

To examine the kinetics of low doses of perfluoro compounds (PFCs), we administered perfluorohexanoic acid (C6A), perfluorooctanoic acid (C8A), perfluorononanoic acid (C9A) and perfluorooctane sulfonate (C8S) with a single oral dose (50-100 µg/kg BW), and in drinking water at 1, 5, and 25 µg/L for one and three months to male rats; and examined the distribution in the brain, heart, liver, spleen, kidney, whole blood and serum. C6A was very rapidly absorbed, distributed and eliminated from the tissues with nearly the same tissue t1/2 of 2-3 hr. Considering serum Vd, and the tissue delivery, C6A was mainly in the serum with the lowest delivery to the brain; and no tissue accumulation was observed in the chronic studies as estimated from the single dose study. For the other PFCs, the body seemed to be an assortment of independent one-compartments with a longer elimination t1/2 for the liver than the serum. The concentration ratio of liver/serum increased gradually from C0 to a steady state. The high binding capacity of plasma protein may be the reason for the unusual kinetics, with only a very small fraction of free PFCs moving gradually to the liver. Although the tissue specific distribution was time dependent and different among the PFCs, the Vd and ke of each tissue were constant throughout the study. The possibility of extremely high C6A accumulation in the human brain and liver was suggested, by comparing the steady state tissue concentration of this study with the human data reported by Pérez et al. (2013).

Sequential transition of the injury phenotype, temperature-dependent survival and transcriptional response in Listeria monocytogenes following lethal H2O2 exposure.

The food-borne pathogen Listeria monocytogenes is present persistently in food processing environments, where this bacterium is exposed to various stress factors, including oxidative stress. This study aimed to elucidate the temperature-dependent response of L. monocytogenes to H2O2 exposure and the phenotypic changes in colony formation by H2O2-treated bacteria. Survival curves indicated an increase in the resistance to H2O2 in L. monocytogenes as the temperature decreased during the stress exposure procedure. Transcriptional induction of genes with key roles in response to H2O2, including sigB and kat, was observed at 37°C, but not at 20°C, whereas other stress response genes were induced at both temperatures. Following H2O2 exposure, L. monocytogenes produced small colony phenotypes and the colony size decreased in a stress exposure duration-dependent manner. Resuscitated cells with no ability to form colonies in the absence of sodium pyruvate were also found. Our findings show the possibility that a sequential transition in the injury phenotype from small colony phenotype to resuscitated cells occurred during the course of exposure to H2O2. The higher H2O2 resistance at 20°C than 37°C suggests further investigation of the response to H2O2 exposure under the lower temperatures, including refrigeration temperature, which may contribute to elucidation of bacterial survival over extended time periods in food-processing environments.

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells.

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.

Characteristics of Listeria monocytogenes isolated from imported meat in Japan.

The genomic structure of the iap region in Listeria monocytogenes (serovar 4b), isolated from chicken imported into Japan, was compared with those from Japanese strains. The isolate was similar to the Japanese strains in a comparatively new, rare group. Such strains might be imported from foreign countries.

Discrimination of Listeria monocytogenes contaminated commercial Japanese meats.

Discrimination was attempted on 14 Listeria monocytogenes strains isolated from commercially available Japanese pork and chicken. Examination of the isolates was performed by restriction fragment length polymorphism (RFLP) analysis of the chromosomal DNA and amplified products and comparison of the nucleotide sequences of the amplified products. A polymorphism region containing the repeated sequences in the iap gene was amplified by the polymerase chain reaction (PCR). The genetic analyses could discriminate the 14 isolates in combination with traditional serotyping, and some strains isolated from different meats were confirmed to have a genetically close relationship. Genetic analyses used in the present study would be useful for the elucidation of the pathogen tracks from contaminated sources to humans and of the ecological niche in the food environment.

Analysis of the molecular evolution of Listeria monocytogenes isolated from Japanese meats and environment.

Food contaminated by Listeria monocytogenes is a problem on a worldwide level because it is a serious food-borne pathogen. Although 3 evolutionary divisions have been reported for L. monocytogenes, the evolution of Japanese isolates has not yet been clarified. Thus, in order to determine the lineage of these Japanese isolates, we classified and conducted phylogenetic analysis of 407 bp (position 1116-1522) of the iap gene derived from 88 isolates from Japanese listeriosis patients, foods and environment. The isolates were classified into 18 types commonly accompanied by serotypes, and the types were divided into 3 lineages. Our results suggest that these Japanese isolates belong to the 3 lineages of L. monocytogenes isolated in other countries.

Thallium contamination in wild ducks in Japan.

Although thallium (Tl) is toxic to both humans and animals, there is little information on contamination in wildlife. In this study, Tl contents in wild ducks in Japan were determined. Contents of Tl in kidney and liver ranged from 0.42 to 119.61 and 0.10 to 33.94 microg/g dry weight, respectively. Significant correlations between Tl contents in kidney and liver were observed for all dabbling ducks except mallard (Anas platyrhynchos); similar correlations were not observed in diving ducks. Variation in Tl content was observed between sampling locations with the highest mean Tl content in the Eurasian wigeon (Anas penelope) collected in Ibaraki Prefecture.

[Infectious disease of simian herpes B virus].

The epidemiology and the method of diagnosis were elucidated for simian herpes B virus (SHBV) infection. It is important that usefulness was demonstrated for the methods of DNA diagnosis having high sensitivity and specificity for SHBV and HSV-1, 2 types, and of detectable serological diagnosis for each specific antibody. The methods allowed the final diagnosis of the human infection due to the reactivation of latent SHBV and the HSV infection from human to monkey. These results would be able to become important and fundamental knowledge for the sero-epidemiological analysis of the infectious stile.

Simultaneous analysis for multiple heavy metals in contaminated biological samples.

In the present study, the conditions of analysis by inductively coupled plasma-atomic emission spectrometry (ICP-AES) were investigated. Twenty-six elements (Mg: 25 ppm; Sc: 10 ppm; Ti: 50 ppm; others: 100 ppm) were used as the elements interfering with selected 24 wavelengths. Consequently, the background values in 19 elements were subjected to some influences. However, all of these effects disappeared at low concentrations--less than 1 ppm of interfering elements. Next, the values from the ordinary calibration method were compared with those from the standard addition method using several biological samples. There was a discrepancy in the results obtained from both methods because of the sample, and three patterns were observed. However, no discrepancy was observed in the values for the standard reference materials using both methods. There was no significant difference between the certified values of the standard reference materials and the obtained ones by ICP. Therefore, the analytical wavelengths and the methods in the present study were suggested to be useful for ICP-AES analysis for environmental and/or biological samples.

Biofilm formation under different temperature conditions by a single genotype of persistent Listeria monocytogenes strains.

Some Listeria monocytogenes strains, termed persistent strains, originate from the same processing plant and have the ability to survive and grow over extended periods of time at contamination sources. In order to evaluate biofilm formation by such persistent strains, we isolated the pathogen from chicken samples collected from the same retail shop in repeated visits over 6 months. Strains that were of serotype 1/2b and were assigned to the same genotype by multi-virulence-locus sequence typing analysis were isolated on repeated occasions from December 1997 to June 1998 and thus were defined as persistent strains. In the present study, biofilm formation by the persistent strains was evaluated using microplates at 30 and 37°C. The biofilm-forming capability was measured after cells attaching to the microplate well were stained with crystal violet. Comparison of biofilm formation at 30°C among the persistent strains showed that a significantly higher amount of the stain was obtained from the persistent strains isolated from December to March than from those isolated from April to June. However, no significant difference in biofilm formation at 30°C was observed between persistent and nonpersistent groups of L. monocytogenes strains. In contrast, biofilm formation at 37°C was consistent among the persistent strains, and they produced significantly more biofilm at 37°C than did the nonpersistent strains. The persistent strains were also found to change their biofilm-forming ability in a temperature-dependent manner, which may suggest that the persistent strains alter their biofilm formation in response to changing environmental factors.