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.

Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

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.

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.

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.

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.

Genetic classification of Listeria monocytogenes serotype 4b strains, including epidemic clones, isolated from retail meat in the Tokyo metropolitan area.

A food-borne pathogen, Listeria monocytogenes serotype 4b, has been frequently isolated from patients with listeriosis, and numerous outbreaks of listeriosis are associated with this serotype. In the present study, we performed subtyping of L. monocytogenes serotype 4b strains on the basis of genetic analyses. Thirty-four isolates of serotype 4b were classified into 8 genotypes, namely genotypes 12, 15, 16, 17, 18, 23, 24, and 25, on the basis of the sequence for the partial iap gene. Genetic analyses revealed that genotype 16 and genotypes 24 and 25 belong to epidemic clone I (ECI) and ECII, respectively, which have been frequently associated with listeriosis outbreaks in the United States and Europe. The genotype isolated most frequently from retail meats in the Tokyo metropolitan area was genotype 12 (52%), followed by genotype 16 (29%), which belongs to ECI. We suggest that ECI is a common subtype of L. monocytogenes in retail meat in the area under investigation. On the other hand, ECII isolates were confirmed to be present in retail meat in Japan but were rare.

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.

Contents of various elements in the organs of seabirds killed by an oil spill around Tsushima Island, Japan.

Twenty nine oil-soaked birds were collected from around the Coast of Tsushima Island. The contents of eight elements in the livers and kidneys of the birds were investigated. Statistically higher concentrations of vanadium and thallium in the liver and of titanium in the kidney were found in the birds that were found dead compared with those that died after rescued. A significant correlation (r=0.695, P<0.01) was observed only for the molybdenum content between the kidneys and livers from the birds found dead. Although the controls of the eight elements of birds investigated in the present study remain unexplained, some of lower concentration in rescued birds can be blamed on a decrease in food intake of birds. The relation between oil contamination and concentration of elements need to be further explored.

Interaction between titanium and cadmium in various guinea pig organs.

Titanium (Ti) is used in many fields, while cadmium (Cd) is known to cause the itai-itai disease. In the present study, possible interactions between titanium and cadmium were investigated. Aorta, taenia coli, and liver were removed from male guinea pigs. Muscle tension was measured using intact aorta and taenia coli and using ß-escin-permeabilized taenia coli in a physiological salt solution and a hyperpotassium solution containing Cd and/or Ti. Cellular Cd contents were determined using all tissues after washout with EDTA solution. Cadmium-induced relaxation in the hyperpotassium solution recovered significantly (P < 0.01) following Ti treatment in taenia coli, but not in the aorta. In ß-escin-permeabilized taenia coli, the percentage recoveries after Cd treatment and after Ti plus Cd treatment were 67.3 ± 8.7 % (n = 4) and 87.7 ± 3.8 % (n = 4), respectively, compared with Ca-induced control contraction. Cellular Cd contents in taenia coli decreased significantly following treatment with Ti 10(-4) M. Although similar results were obtained using the aorta and the liver, there were no significant differences between the control and Ti 10(-5) M. High concentrations of Ti may reduce cellular Cd content.

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 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.

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.

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.

Spectrum of Rift Valley fever virus transmission in Kenya: insights from three distinct regions.

Rift Valley fever virus (RVFV) is an emerging pathogen that maintains high biodefense priority based on its threat to livestock, its ability to cause human hemorrhagic fever, and its potential for aerosol spread. To define the range of human transmission during inter-epidemic and epidemic periods in Kenya, we tested archived sera from defined populations (N = 1,263) for anti-RVFV IgG by ELISA and plaque reduction neutralization testing. RVFV seroprevalence was 10.8% overall and varied significantly by location, sex, and age. In NW Kenya, high seroprevalence among those born before 1980 indicates that an undetected epidemic may have occurred then. Seroconversion documented in highland areas suggests previously unsuspected inter-epidemic transmission. RVFV seroprevalence is strikingly high in certain Kenyan areas, suggesting endemic transmission patterns that may preclude accurate estimation of regional acute outbreak incidence. The extent of both epidemic and inter-epidemic RVFV transmission in Kenya is greater than previously documented.

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.

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.

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR.

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

Specific detection and identification of herpes B virus by a PCR-microplate hybridization assay.

Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined.