Effect of climatic elements on Campylobacter colonization in broiler flocks reared in southern Japan from 2008 to 2012.

To demonstrate the effect of climatic elements on Campylobacter colonization in broiler chickens reared in Japan, the correlation between Campylobacter isolated from chickens (191 of 236 flocks, 80.9%) between 2008 and 2012 and climatic elements was analyzed by logistic regression. We divided the rearing process into 13 terms of 5 d each (total: 65 d). Terms were numbered backwards, wherein a 0-term lag was considered as the sampling day plus 4 d before sampling; 1-term lag was the 5-d term before the 0-term lag, and so on, until the 12-term lag. We obtained climatic data tracing back from the 0-term to the 12-term lags. For evaluation in each season, we divided chickens reared during periods of rising temperature (spring, summer) and decreasing temperature (autumn, winter). Air temperature showed a positive correlation with Campylobacter colonization from the 0- to 12-term lags in chickens reared during the period of rising temperature (odds ratio [OR], 1.069 to 1.104), and from the 0- to 4- and 6-term lags (OR, 1.079 to 1.105) in chickens reared during the period of decreasing temperature. The strong positive effect of air temperature on Campylobacter colonization, particularly during the period of rising temperature, may be associated with the effect on the Campylobacter environmental sources and/or vectors. A positive correlation was observed between Campylobacter colonization and humidity when chicken houses were empty and new chicks were introduced (from the 9- to 12-term lags) during the period of decreasing temperature (OR, 1.076 to 1.141). Thus, high humidity would be an important factor causing carry-over of Campylobacter infection during the period of decreasing temperature. We also found that solar radiation increased Campylobacter colonization during the period of decreasing temperature, from the 2- to 8-term lags, except for the 4- and 5-term lags, in Japan. The results of this study demonstrate the effects of air temperature, humidity, and solar radiation on Campylobacter colonization in broiler chickens, and are potentially important for developing strategies to reduce the risk of Campylobacter contamination in broiler chickens.

Distribution of extended-spectrum cephalosporin resistance determinants in Salmonella enterica and Escherichia coli isolated from broilers in southern Japan.

This study was conducted to investigate the distribution and diversity of extended-spectrum cephalosporin (ESC) resistance determinants in Salmonella enterica and Escherichia coli obtained from the same cecal samples and to provide evidence of transmission of the resistance determinants among these bacteria in broiler farms in southern Japan. Salmonella enterica and E. coli were characterized by serotyping and multilocus sequence typing, respectively. An antimicrobial susceptibility test, plasmid analysis, and identification and localization of resistance genes were performed to determine the relatedness of ESC resistance determinants among the isolates. Of 48 flocks examined, 14 had S. enterica. In total, 57 S. enterica isolates were obtained, 45 of which showed ESC resistance. Extended-spectrum cephalosporin-resistant E. coli were also obtained from all of these ESC-resistant Salmonella-positive samples. ß-Lactamase genes, blaTEM-52 (38 isolates), blaCTX-M-14 (1 isolate), and blaCMY-2 (6 isolates), were carried by conjugative untypable or IncP plasmids detected in the S. enterica serovars Infantis and Manhattan. The ß-lactamase genes blaCTX-M-14 (3 isolates), blaCTX-M-15 (3 isolates), blaSHV-2 (1 isolate), blaSHV-12 (2 isolates), and blaCMY-2 (32 isolates) associated with IncI1-Iγ, IncFIB, IncFIC, IncK, IncB/O, and IncY plasmids were detected in E. coli co-isolates. Restriction mapping revealed similar plasmids in Salmonella Infantis and Salmonella Manhattan and in different sequence types of E. coli. Intraspecies transmission of plasmids was suggested within S. enterica and E. coli populations, whereas interspecies transmission was not observed. This study highlights the importance of plasmids as carriers of ESC resistance determinants.

Natural contamination of dietary rice straw with zearalenone and urinary zearalenone concentrations in a cattle herd.

The present study was conducted to 1) identify the natural source of feed contamination by zearalenone (ZEN), which was suspected to have caused persistently increased urinary ZEN concentrations in one of our experimental cattle herds, and 2) evaluate the effects of intervention against this source of contamination. As an experimental model, a fattening Japanese Black cattle herd showing persistently increased urinary ZEN concentrations was identified. Urinary ZEN concentrations of cows fed with new rice straw (experimental group, n = 6) vs. cows that continued to feed on the old rice straw (control group, n = 4) were measured at the start (d 1) and at 2 wk (d 14) after the onset of feeding with straw. In addition, the ZEN concentration in feed and water samples was measured by using both the ELISA and HPLC methods. Furthermore, isolation and identification of fungi from rice straw and concentrate feed samples were performed. The urinary ZEN concentration [ZEN (pg/mL)/creatinine (mg/mL) = pg/mg of creatinine] of cows fed with new rice straw was significantly (P < 0.05) less (843 pg/mg of creatinine) than that of cows fed with old rice straw (15,951 pg/mg of creatinine). On both d 1 and 14, the ZEN concentrations of old rice straw were greater than those of new rice straw. In addition, fungal colonies were observed in the culture media that was obtained from the old rice straw suspected of ZEN contamination, but not in the culture media from new rice straw or other feed samples. In conclusion, our field trials clearly indicate that the rice straw fed to the cows was naturally contaminated with ZEN, and that the monitoring of urinary ZEN concentrations could prove to be a useful tool for detecting the exposure of cattle to ZEN contamination at the farm level.

Temporal distribution and genetic fingerprinting of Salmonella in broiler flocks from southern Japan.

During the 1998 to 2003 period, cecal contents of 4,024 broiler chickens from 252 flocks raised in 63 holdings were examined for Salmonella. The aims were to establish the actual status of the infection, its temporal distribution, prevalent serotype, and common genotype among broiler flocks brought at the slaughterhouse. Collected samples were preenriched in Hajna tetrathionate broth, and after 24 h of incubation, 10 microL of the broth was streaked on selective Rambach agar plate. Suspected scarlet color colonies of Salmonella were cloned on nutrient agar, confirmed through biochemical tests and sero-typed using O and H antigens. Pulsed field gel electrophoresis technique generated DNA fragments banding patterns and established their clonal relatedness. Salmonella was isolated from 563 (14%) samples in 179 (71%) flocks. The flock situation varied from Salmonella-negative holdings (n = 9), positive-flocks from persistently infected holdings (n = 21), and holdings (n = 19) that showed fluctuations with alternating negative and positive flocks for variable time periods. Fourteen holdings (negative, n = 5 and positive, n = 9) were sampled once throughout the study period. Seasonality component was not observed, and salmonellae were found colonizing broiler ceca in warm and cold months. Predominant serovar was Salmonella Infantis (93.3%; n = 525). Macrorestriction fingerprints of Salmonella Infantis using XbaI presumed the isolates to be derived from a common parent. Enhanced discrimination by BlnI digestion produced 3 banding patterns that were closely related genetically and hence epidemiologically related. Such epidemiological information may enable producers to formulate effective control action plan tailored for individual holdings with special emphasis on biosecurity, hygiene, and pest control.

Motor learning of hands with auditory cue in patients with Parkinson's disease.

In the present research, changes in motor cortex function were observed in relation to repetitive, voluntary thumb movement (training) in patients with Parkinson's disease (PD) and normal control subjects. Changes in the direction of thumb movement due to motor evoked potential (MEP) by transcranial magnetic stimulation (TMS), after motor training with and without rhythmic sound, were measured using a strain gauge for 12 patients with PD and 9 normal control subjects. PD patients who experienced the freezing phenomena showed poor change in direction of TMS-induced movement after self-paced movement; however, marked change in direction of TMS-induced movement was observed after training with auditory cue. PD patients who had not experienced the freezing phenomena showed positive effects with the auditory cue, producing similar results as the normal control subjects. Two routes for voluntary movement are available in the nervous system. The decreased function of basal ganglia due to PD impaired the route from the basal ganglia to the supplementary motor cortex. These data suggest that the route from sensory input to cerebellum to premotor cortex could compensate for the decreased function of the route via the basal ganglia to the premotor cortex. Once change in the motor cortex occurred, such change persisted even after the interruption of training. These phenomena suggest that motor memory can be stored in the motor cortex.

Simultaneous detection of mutations associated with resistance to macrolides and quinolones in Campylobacter jejuni and C. coli using a PCR-line probe assay.

Quinolone and macrolide resistance of Campylobacter jejuni and Campylobacter coli mainly depend on a mutation in gyrA and in 23S rDNA, respectively. In order to detect quinolone and/or macrolide resistant C. jejuni and C. coli strains, a macrolide and quinolone line probe assay (MQ-LiPA) was developed and 42 C. jejuni and C. coli strains were tested to evaluate the efficiency of the assay. Profiles of the mutations in 23S rDNA and in gyrA characterized by MQ-LiPA agreed with resistance to macrolides and quinolones. MQ-LiPA is a rapid and simple method for simultaneous detection of quinolone and macrolide resistance of C. jejuni and C. coli. We could also discriminate between C. jejuni and C. coli using probes for detection of gyrA mutations in MQ-LiPA.

Cortical reorganization in training.

Plasticity within the human central motor system occurs and has been studied with transcranial magnetic stimulation in patients with amputations, spinal cord injuries, and ischemic nerve block. These studies have identified a pattern of motor system reorganization that results in enlarged muscle representation areas and large motor evoked potentials (MEPs) for muscles immediately proximal to the lesion. Some of these changes are apparent minutes after ischemic nerve block, weeks after spinal cord injury, and as early as six months after amputation.These studies motivated us to study the cortical motor reorganization after finger movement training in normals and after anastomosis of intercostal nerves to the musculocutaneous nerve in young patients with cervical root avulsions due to a traumatic motorcycle injury.

Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay.

Mutation of 23S rDNA is one of the mechanisms of erythromycin resistance. PCR and line probe assay (PCR-LiPA) with ten oligonucleotide probes were developed to detect the mutations associated with macrolide resistance at positions of 2072, 2073 and 2074 in 23S rDNA of Campylobacter jejuni/coli. A2074-->G mutation was detected in 12 of 25 isolates, which were resistant to erythromycin. No other mutations in 23S rDNA were detected. The rest of the strains were susceptible to erythromycin and no mutation in 23S rDNA was detected. Six laboratory induced erythromycin resistant mutants had no mutations in 23S rDNA. PCR-LiPA is a useful and rapid method to detect mutations in 23S rDNA associated with erythromycin resistance in C. jejuni/coli.

Antimicrobial susceptibilities of Campylobacter strains isolated from broilers in the southern part of Japan from 1995 to 1999.

Cecal contents (16 samples/each flock) of broilers derived from 212 flocks were investigated for colonization of Campylobacter from 1995 to 1999 in the southern part of Japan, and the isolates were tested for antimicrobial susceptibilities. C. jejuni-positive flocks numbered 42 (19.8%) and C. coli-positive ones 26 (12.3%); Campylobacter spp. were recovered from 68 flocks (32.1%) in total. MICs of ampicillin, erythromycin (EM), tetracycline, nalidixic acid (NA), norfloxacin (NFLX), and ofloxacin (OFLX) to these 68 Campylobacter isolates were determined. Quinolone-resistant Campylobacter isolates numbered 22 (32.4%). All the isolates except one were cross-resistant to NA, OFLX, and NFLX. A high frequency of quinolone-resistance was found in both C. jejuni and C. coli, whereas a high level of EM-resistance was found in only C. coli strains. All C. jejuni isolates were sensitive to EM.

A mitochondrial encephalo-myo-neuropathy with a nucleotide position 3271 (T-C) point mutation in the mitochondrial DNA.

We report three members of a family, who exhibited a phenotype similar to 'myoclonus epilepsy with ragged-red fibers' but had a genotype usually associated with 'mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes'. The patients, a 48-year-old female, and her two sons, aged 21 and 19 respectively, presented with photo-reactive syncopal episodes, disturbances of gait and writing, dysarthria and finger tremor since the 3rd and 2nd decade of life, respectively, that were accompanied also by numbness and weakness of the extremities. Subsequently, cerebellar ataxia and myoclonus were also noted. Electromyography revealed both myogenic and neurogenic muscular changes, and nerve conduction studies demonstrated a sensory-motor neuropathy. Biopsy showed ragged-red fibers with strongly stained SDH-positive vessels in skeletal muscles, and a marked loss of myelinated fibers of the sural nerves. Mitochondrial (mt) DNA analyses of peripheral blood, muscles and nerves revealed that all members had a heteroplasmic np3271 (T-C) point mutation in the mitochondrial tRNA-Leu gene (UUR). This family is unique, in that all patients presented with a myoclonus epilepsy with ragged-red fibers-like phenotype and had a distinctive peripheral neuropathy, while the detected mtDNA 327l (T-C) mutation has been reported to date only in rare cases of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes

beta-Phenylethylamine modulates acetylcholine release in the rat striatum: involvement of a dopamine D(2) receptor mechanism.

We examined the effects of beta-phenylethylamine on striatal acetylcholine release in freely moving rats using in vivo microdialysis. beta-Phenylethylamine at 12.5 mg/kg, i.p. did not affect acetylcholine release in the striatum, whereas 25 and 50 mg/kg, i.p. immediately induced an increase in acetylcholine release in the striatum at 15-45 min. This increase following intraperitoneal administration of beta-phenylethylamine (25 mg/kg) was not affected by locally applied SCH-23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine, 10 microM), a dopamine D(1) receptor antagonist, nor by raclopride (10 microM), a dopamine D(2) receptor antagonist. The increased release of acetylcholine induced by beta-phenylethylamine was suppressed by local infusion of tetrodotoxin (1 microM). In contrast, the extracellular acetylcholine level in the striatum was significantly decreased by local application of beta-phenylethylamine (10 and 100 microM) in the striatum via a microdialysis probe. The decrease was completely blocked by local co-application of raclopride (10 microM). The beta-phenylethylamine-induced decrease in striatal acetylcholine release was not affected by co-perfusion with SCH-23390 (10 microM). These results indicate that systemic administration of beta-phenylethylamine increases acetylcholine release, whereas locally applied beta-phenylethylamine decreases striatal acetylcholine release in freely moving rats. Furthermore, the dopaminergic system, through the dopamine D(2) receptor, is involved in the locally applied beta-phenylethylamine-induced decrease in acetylcholine in the striatum.

Oculopharyngeal muscular dystrophy in a Japanese family with a short GCG expansion (GCG)(11) in PABP2 gene.

Clinicopathological and molecular genetic findings on a new Japanese family with oculopharyngeal muscular dystrophy are reported. The family has 54 members, ten of whom are affected (seven male and three female), in 3 generations. Three affected males, one affected female and one unaffected female of seven living siblings in the third generation were examined. Bilateral ptosis developed in the 4th and 5th decades in the three male cases, and in the 7th decade in the female, and this was followed by diplopia, nasal voice, dysphagia and muscle weakness. In addition, severe external ophthalmoplegia, dysphonia, and proximal amyotrophy were prominent in this family. Electromyographs revealed myogenic/neurogenic changes, and computed tomography disclosed selective muscle wasting with fatty replacement, predominantly in the lower extremities. Muscle biopsy in the four affected patients showed variation in fiber size, and the presence of small angulated fibers and occasional rimmed vacuoles. Electron microscopic examination revealed an accumulation of filamentous inclusions in muscle fiber nuclei. DNA analysis identified that (GCG)(6) in the PABP2 gene was expanded to (GCG)(11) in the four affected cases examined. All studies were negative in the one unaffected. These results confirm that OPMD is caused by GCG short expansion and provides insights into the genetic mechanisms which may contribute to adult onset myopathy, confined to oculopharyngeal muscles.

[Two cases of cranial pachymeningitis presenting only severe continuous headache or convulsion].

In cranial pachymeningitis, headache, cranial nerve palsies, and ataxia were the most common clinical manifestations. We reported two rare cases presenting only headache or convulsion as a main symptom. A 66-year-old man was suffering from severe continuous headache for years with no other clinical symptoms. Laboratory data revealed positive RA test and elevated RAPA. With oral steroid therapy, his headache was subsided. A 39-year-old woman visited us with convulsive attacks as only symptom. Biopsy of the thickened dura revealed granulated epithelioid cells without central necrosis. With antituberculosis therapy, hypertrophy of dura, as well as the symptom, was disappeared. It is difficult to diagnose pachymeningitis when patients manifest only headache or convulsion. We suggest to carry out Gd-enhanced MR scan in these cases.

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter.

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.

The effect of methamphetamine on the release of acetylcholine in the rat striatum.

We examined the effect of methamphetamine on the release of acetylcholine in the striatum of freely moving rats, using an in vivo microdialysis method. The basal level of acetylcholine was 3.67+/-0.47 pmol/30 microl per 15 min in the presence of neostigmine (10 microM). Tetrodotoxin (1 microM), a selective blocker of voltage-dependent Na+ channels, markedly inhibited the release of acetylcholine in the striatal perfusates. Apomorphine (1.0 mg/kg, i.p.), a dopamine receptor agonist, also significantly attenuated acetylcholine release. Methamphetamine (0.1 and 0.5 mg/kg, i.p.) did not immediately affect acetylcholine release in the striatum, but a dose of 1.0 mg/kg (i.p.) induced an increase of acetylcholine release in the striatum at 15-60 min. Striatal infusion of methamphetamine (5 and 10 microM) did not influence acetylcholine release. The increase following intraperitoneal administration of methamphetamine was slightly diminished by haloperidol (0.5 mg/kg). After microinjection of the neurotoxin, 6-hydroxydopamine (6 microg/3 microl), in the substantia nigra 7 days before, the increase of acetylcholine induced by the administration of methamphetamine (1.0 mg/kg) was slightly attenuated, whereas the administration of reserpine (2 mg/kg, i.p.) 24 h before, combined with alpha-methyl-p-tyrosine (300 mg/kg, i.p.) 2.5 h before, completely blocked the increase in release of acetylcholine. These findings suggest that methamphetamine exerts an excitatory influence on striatal acetylcholine release in freely moving rats, and that this excitatory effect involves the dopaminergic system and the catecholaminergic system.

Analysis of distribution of Campylobacter jejuni and Campylobacter coli in broilers by using restriction fragment length polymorphism of flagellin gene.

The incidence of Campylobacter jejuni and Campylobacter coli in broiler farms was 33.9% (19/56). C. jejuni-positive flocks accounted for 20.0% (17/85) and C. coli-positive ones was 4.7% (4/85). There were 14 patterns (fla type) of restriction fragment length polymorphism (RFLP) of flagellin A gene among these 22 strains of C. jejuni and C. coli including the standard strain C. jejuni ATCC 33560. Different fla types of Campylobacter were isolated from broilers in different growing cycles on the same farms. Four strains of C. jejuni were isolated from four breeder farms and four fla types of C. jejuni were detected from their progenies reared on growing farms. Three fla types of C. jejuni detected from the progenies were different from those of each breeder. Also, the other three fla types of C. jejuni were detected from different progenies of each growing farm during the next growing cycle. These findings indicate that the RFLP analysis may contribute to epidemiological studies of C. jejuni and C. coli contamination of broilers and suggest the risk of contamination with different types of Campylobacter in every growing cycle of broilers on the farm even on the same farm. They also supported that there was little likeliness of the vertical transmission of C. jejuni and C. coli from breeders to broilers.

Direct detection of Campylobacter jejuni in chicken cecal contents by PCR.

Campylobacter jejuni in chicken feces was detected by PCR and Southern blot hybridization (SBH). The detection limits of C. jejuni in chicken feces were 34,000 cells by PCR and 340 cells by SBH. Some cecal contents of chickens up to 3 weeks old were C. jejuni positive by SBH whereas all of them were negative by PCR. Two of 51 cecal contents of 18-day-old chicken embryos were C. jejuni positive by PCR and SBH; but, C. jejuni were not isolated from the samples by conventional culture with selective enrichment.

The relationship between trinucleotide repeat length and phenotypic variation in Machado-Joseph disease.

Trinucleotide repeat expansion in the Machado-Joseph disease (MJD) gene has been found in 26 patients from 20 unrelated Japanese families. Expanded alleles had 68 to 84 repeats, whereas normal alleles had 14 to 37 repeats. The age of onset was inversely correlated with the repeat length. To evaluate in detail the relationship between the repeat length and clinical features, we subdivided the 26 patients into three groups on the basis of the repeat length (group 1, 78 repeats or more; group 2, 74 to 77 repeats; group 3, 73 repeats or less). Group 1 and group 2 had common features of spasticity, hyperreflexia, Babinski sign, bulging eyes, facial myokymia and extrapyramidal signs as well as cerebellar ataxia and ophthalmoplegia. It should be noted that group 1 showed more prominent pyramidal and extrapyramidal signs than group 2. In contrast, group 3 showed hypotonia, hyporeflexia and sensory disturbance in addition to cerebellar ataxia and ophthalmoplegia. These findings suggest that the repeat length plays an important role in phenotypic variation. DNA analysis for the MJD mutation was clearly useful for making an accurate diagnosis in patients without bulging eyes, facial myokymia, dystonia or marked spasticity.