Aberrant forms of Yersinia pseudotuberculosis as spheroplasts and filaments in yersiniosis in squirrel monkeys.

This report describes atypical cases of yersiniosis in squirrel monkeys in which aberrant forms of Yersinia pseudotuberculosis were seen. There were 2 outbreaks due to yersiniosis in squirrel monkeys in Japan. The monkeys had systemic necrotizing and hemorrhagic lesions with Gram-negative rod-shaped bacilli and microthromboembolism in the kidneys. Some lesions contained filaments, globular bodies, and other pleomorphic forms of bacteria. All forms were usually seen in the same lesions, and those with pleomorphic morphology appeared to be an intermediate form between the rod-shaped bacteria and the filaments or globular bodies. In addition, they had strong immunolabeling for Y. pseudotuberculosis, as did the rod-shaped bacteria. Therefore, the globular bodies, filaments, and others are strongly suspected to be shape-changed bacilli of Y. pseudotuberculosis. These morphologically altered bacteria could cause errors in diagnosis since they resemble fungi or protozoa, and special staining techniques, including immunohistochemistry, can be helpful in establishing the correct diagnosis.

A novel multiplex PCR assay for Salmonella subspecies identification.

AIM: To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification. METHODS AND RESULTS: Five primer pairs were chosen to detect the genes (fljB, mdcA, gatD, stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella. The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non-Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94.3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay. CONCLUSIONS: The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.

Real-time PCR method for quantification of Staphylococcus aureus in milk.

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 10(7) to 10(1) CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 10(7) to 10(1) CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63 degrees C for 30 min (low-temperature long-time method) but not at 72 degrees C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

Fatal Salmonellosis in Captive Maras (Dolichotis patagonum) Caused by Salmonella Enteritidis.

We report epidemic occurrences of fatal salmonellosis caused by Salmonella Enteritidis in captive maras (Dolichotis patagonum) in a zoological garden in Japan. The deaths were sudden or followed a peracute course within a few hours of the first observations of clinical abnormalities. Gross lesions included haemorrhages in the subcutis and skeletal muscles, liver, spleen, lung, heart and gastrointestinal tract. Microscopically, there were haemorrhagic and necrotizing lesions with gram-negative bacilli in the liver, spleen, small intestine and Peyer's patches. These bacilli showed strong immunolabelling for Salmonella O9 antigen and S. Enteritidis was isolated from the lesions. To the best of our knowledge, this is the first report of salmonellosis in maras.

Differences in heat resistance among pathogenic Yersinia enterocolitica depended on growth temperature and serotype.

To gain a better understanding about the effect of growth temperature on heat resistance of Yersinia enterocolitica, we determined decimal reduction times at 60 degrees C (D60-values) for O:3; O:5,27; O:8; and O:9 strains harboring virulence plasmid coding for Yersinia outer membrane protein and experimentally virulence plasmid-deleted strains after they were grown to stationary phase at 7, 25, or 37 degrees C. Bacteria were inoculated into Trypticase soy broth and were incubated at several temperatures. D60-values of O:3; O:5,27; and O:8 strains were larger when they were grown at 37 degrees C than at 7 or 25 degrees C, despite the presence or absence of virulence plasmids. However, similar D60-values were observed in O:9 strains, despite growth at 7, 25, or 37 degrees C. The results indicate two types of Y. enterocolitica strains, growth temperature-dependent and -independent, and a Yersinia outer membrane protein that is not directly involved in growth temperature-dependent heat resistance.

Experimental infection with Yersinia enterocolitica serovar 0:8 in beagle dogs.

Dogs were challenged orally with Yersinia enterocolitica serovar 0:8 biovar 1 for assessment of the infectivity of 0:8 bacteria. The bacteria were shed in the feces for 7-21 days following oral challenge. They were also recovered from intestinal contents and small intestinal Peyer's patches, but not from deeper organs of dogs euthanized 3 and 7 days after oral challenge. Dogs challenged subsequently with 10(10) bacteria showed protection from establishment of the bacteria in the intestinal tract. High titers of serum O-agglutinins developed in the dogs challenged with the bacteria. No clinical or hematological abnormalities were observed. The possibility that dogs may be a source of infection of 0:8 bacteria to human is discussed.

Occurrence of Erysipelothrix spp. in chicken meat parts from a processing plant.

From March 1996 to March 1997, 153 domestic raw chicken meat samples, including 71 thigh, 50 outer breast muscle, and 32 white meat samples, from a processing plant located in a chicken abattoir in Nagano Prefecture, Japan, were examined for the presence of Erysipelothrix spp. Erysipelothrix spp. were isolated from 49 (30.0%) of the 153 chicken meat samples. Of 67 Erysipelothrix isolates, 65 and 2 isolates were identified as E rhusiopathiae and E. tonsillarum. E. rhusiopathiae and E. tonsillarum isolates were serotyped into 11 and 2 different serovars, respectively. These findings might indicate that domestic chicken meat is frequently contaminated with E. rhusiopathiae and seems to be a potential source of human Erysipelothrix infection.

Occurrence of Erysipelothrix spp. in broiler chickens at an abattoir.

From September 1995 to August 1996, 750 chickens from 66 farms sent to an abattoir in Nagano Prefecture, Japan, were examined for the presence of Erysipelothrix spp. Erysipelothrix spp. were isolated from 118 (15.7%) of 750 skin samples, 27 (7.3%) of 372 hypoderm samples, 12 (1.9%) of 630 throat samples, 106 (59.2%) of 179 feather samples, and none of 257 spleen samples. Of 66 farms, 55 farms (83.3%) sent Erysipelothrix-positive chickens and 11 farms (16.7%) only negative ones. Of 297 Erysipelothrix isolates, 273 isolates were identified as Erysipelothrix rhusiopathiae and 24 as Erysipelothrix tonsillarum. E. rhusiopathiae isolates were serotyped into nine different serovars. Of the 273 E. rhusiopathiae isolates, 33 (11.1%) were serotyped to serovar 6; 22 (7.4%) were serovar 5; 19 (6.4%) were serovar 2; 15 (5.1%) were serovar 8; 2 (0.7%) were serovar 21; 4 each (1.3% each) were serovars 1b, 9, 12, and 19; and 178 (59.9%) were untypeable. Of 24 E. tonsillarum isolates, 15 (5.1%) were serotyped to serovar 3, and 9 (3.0%) were serovar 7. These findings indicate that chickens seem to be a potential reservoir of Erysipelothrix spp. in nature and to be a source of human Erysipelothrix infection.

Bacterial contamination in the environment of food factories processing ready-to-eat fresh vegetables.

A total of 196 samples were collected from equipment for trimming, washing, slicing, soaking, dehydrating, blending, and packaging and from the floor and air of operation rooms before and after operation in two food factories processing ready-to-eat fresh vegetables located in the suburbs of Tokyo. Heavy contamination determined by an aerobic plate count of >5.0 log CFU/cm2 or ml was observed after operation in most of the samples examined, as were samples taken before operation on the interior surfaces of equipment for washing, slicing, dehydrating, and blending, the surfaces of blades for slicing, and the floor surfaces of operation rooms. From these environmental samples, the coliform group was detected before operation. Although 67 strains of 70 coliforms isolated were nonfecal, three Escherichia coli strains were detected in the surface of the operation room floors and the gloves of employees. Bacillus cereus was isolated from 9 of 86 and 17 of 85 samples examined before and after operation with the number of 2.0 to 3.0 log CFU/cm2 or ml. Listeria spp. were not detected in the environment of the food factories.

Bacterial contamination of ready-to-eat foods and fresh products in retail shops and food factories.

Raw vegetables cut for salad, cooked salad, cooked rice, boiled noodles, bean curd, and cooked Japanese foods were purchased in 27 retail shops in Tokyo. Intact vegetables before being processed and ready-to-eat fresh salad products were obtained from two food factories located in the suburbs of Tokyo. Two hundred thirty-eight retail samples, 137 samples of intact vegetables, and 159 samples of fresh products were examined for aerobic plate count (APC), coliforms, Escherichia coli, Listeria spp., Staphylococcus aureus, and Bacillus cereus. The APC of retail foods were 2.1 to 5.7 log CFU/g, and the range for the coliforms was 0.1 to 2.3 log CFU/g. The APC and coliform values showed that the raw vegetables cut for salad were the most heavily contaminated among the six kinds of ready-to-eat foods examined. Although L. monocytogenes was not detected, two samples of raw vegetables and five kinds of cooked foods yielded Listeria spp. S. aureus was detected in one sample of Japanese cooked food. The APC of the intact vegetables were 2.9 to 7.3 log CFU/g upon arrival and 2.2 to 7.2 log CFU/g after 3 days storage at 10 degrees C. The APC of the fresh products were 3.4 to 7.6 log CFU/g upon arrival and 4.7 to 8.7 log CFU/g after 3 days storage at 10 degrees C. The isolation rates for coliforms were 6.1 to 50% for intact vegetables and 50 to 66.7% for fresh products. E. coli was detected only in the fresh products. B. cereus was isolated from 20.1% (17 of 81) of the intact vegetables and 9.2% (8 of 87) of the fresh products.

Contamination of Salmonella blockley in the environment of a poultry farm.

Cecal and environmental samples were collected and examined for the presence of Salmonella on a farm where a high prevalence of Salmonella blockley in chickens was observed. Of 895 cecal and 525 environmental samples examined, 242 (27.0%) and 202 (38.5%) samples, respectively, yielded S. blockley. From the analysis of plasmid profile patterns, 11 different plasmid profile patterns were found among 444 isolates, with plasmid patterns c and d the most frequent among the isolates from ceca and the environment. Salmonella blockley was isolated from environmental samples such as floor litter, walls, drinking water, waste water, dust, and soil collected when barns were occupied and was positive in drinking water, waste water, and soil when samples were collected from empty barns with occupied neighboring barns, but it was negative in all environmental samples with the exception of soil when the environmental samples were collected from empty barns with empty neighboring barns.

Morphology of intestinal colonization of Yersinia enterocolitica serovar O3 in mice.

The present study was made to know the morphology of the initial invasion and lesions involved in the intestinal colonization of Yersinia enterocolitica serovar O3 in the epithelium of Peyer's patches of mice. Microfold (M) cells formed a specific structure like a pseudopodium and the bacteria were observed on the surface of the pseudopodium-like structure 4 hr after oral administration of serovar O3. The colonies of serovar O3 were observed in the epithelium and the lamina propria of the Peyer's patches dome region, and the bacteria grown in the Peyer's patches were in direct contact with the lumen without covered with the host tissue 24 hr after the administration.

Bacterial flora of the respiratory tracts in chickens with a particular reference to Lactobacillus species.

The effects of three different types of breeding such as isolator, floor, and cage breedings on the bacterial flora of the respiratory tracts (nasal cavity, tongue, pharygolarynx, trachea and air sac) in chickens were determined. Total viable bacterial numbers on the nasal mucus of chickens in the isolator breeding as control group (Group A) aged of 14 days were 10(4.6)/g of autopsy specimen (wet weight), 10(5.7)/g of sample in the cage breeding (Group B) aged of 28 days, and 10(7.0)/g of sample in the floor breeding (Group C) aged of 28 days. Staphylococci and micrococci were predominant bacteria in the nasal cavities of all groups. Total viable numbers of tongue and pharygolarynx were from 10(5.4) to 10(6.5)/g of autopsy specimen. Lactobacilli were the predominant bacteria in pharyngolarynx of chickens. The incidence of staphylococci and micrococci in trachea was lower than those in the another regions. Staphylococci and micrococci dominated in the air sacs of two groups (B and C), but the number and incidence of lactobacilli in the air sacs of chickens were lower than those in the another respiratory tracts. The only clostridia isolation in the air sacs of Group A was observed. A total of 75 strains of Lactobacillus species was isoalted from all respiratory organs and intestine of chickens. These strains were divided into 19 groups. Lactobacillus salivearius subsp. salivarius was the predominant lactobacilli isolated from tongue and pharyngolarynx. Most of isolates from the chicken intestines were mainly identified as the L. acidophilus group and L. reuteri.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence and persistence of Salmonella in broiler chicken flocks.

Cecal contents of 2,345 broiler chickens consisting of 28 flocks originated from 12 farms were examined for the prevalence of Salmonella to know the actual status of infection with Salmonella in the chicken flocks. Salmonella was isolated from 336 (14.3%) samples. From these isolates, eight serovars were identified. Of the 336 Salmonella isolates, 242 (72.0%) were serotyped as S. Blockley, 60 (17.9%) S. Hadar, 15 (4.5%) S. Bredeney, nine (2.7%) S. Schwarzengrund, four (1.2%) S. Anatum, three (0.9%) S. Enteritidis, two (0.6%) S. Ohio, and one (0.3%) S. Livingstone. The same serovars of Salmonella were repeatedly found in the chickens from the same farms. S. Typhimurium and S. Enteritidis were detected in pooled broken eggshell samples collected from the hatchery. Analysis of plasmid profiles revealed 11 patterns of S. Blockley and seven patterns of S. Hadar. Strains of the same plasmid profiles of S. Blockley were isolated repeatedly from the same farm over one year after the first isolation.

A survey for Yersinia pseudotuberculosis in migratory birds in coastal Japan.

Yersinia pseudotuberculosis was isolated from three specimens of two species of birds, the black-faced bunting (Emberiza spodocephala) and pied wagtail (Motacilla alba), of 528 specimens of birds examined from coastal regions in Japan. The two isolated strains of Y. pseudotuberculosis were identified as serovar 4b and serovar 3. This is the first isolation of Y. pseudotuberculosis from birds in Japan. Yersinia enterocolitica was isolated from three specimens of the pied wagtail, one specimen of the reed bunting (Emberiza schoeniclus) and one specimen of the rustic bunting (Emberiza rustica). Yersinia frederiksenii was isolated from two specimens of the gray-rumped sandpiper (Heteroscelus brevipes). Yersinia intermedia was isolated from one specimen of the pied wagtail.

Occurrence of Yersinia enterocolitica in the Tokyo Tama Zoo.

Yersinia enterocolitica serogroups O5A and O8 were isolated from fecal samples of one colony of Japanese macaques (Macaca fuscata) from the Tokyo Tama Zoo (Japan). Serogroup O5A was detected in brown bear (Ursus arctos) prior to the isolation from the macaques. Serogroup O5A organisms also were isolated from the Japanese macaques' breeding area. Serogroup O5A and O8 isolates were not pathogenic. Serogroup O8 isolates did not possess the O7 or O19 antigens.

Outbreak of yersiniosis in Egyptian rousette bats (Rousettus aegyptiacus) caused by Yersinia pseudotuberculosis serotype 4b.

This report describes an outbreak of yersiniosis in Egyptian rousette bats (Rousettus aegyptiacus) caused by Yersinia pseudotuberculosis serotype 4b. Twelve of 61 bats died between November and December 2008 or in May 2009. The bats often displayed multiple yellow-white nodules in the spleen and liver. Microscopically, these consisted of focal necrosis accompanied by inflammatory cell infiltration and colonies of gram-negative bacilli. The bacterial colonies were identified immunohistochemically as Y. pseudotuberculosis O4 and Y. pseudotuberculosis serotype 4b was identified by bacteriological examination. Polymerase chain reaction demonstrated that the isolate harboured the virulence genes virF, inv and ypmA. YPMa is as a superantigenic toxin that is associated with acute systemic infection in man and may contribute to the virulence of Y. pseudotuberculosis in bats.

Prevalence of Salmonella, Yersinia and Campylobacter spp. in feral raccoons (Procyon lotor) and masked palm civets (Paguma larvata) in Japan.

To estimate the public and animal health risk that alien species pose, the prevalence of Salmonella, Yersinia, and Campylobacter spp. in feral raccoons (Procyon lotor, n=459) and masked palm civets (Paguma larvata, n=153), which are abundant alien species in Japan, was investigated in urban and suburban areas of Japan. Salmonella enterica was detected from 29 samples [26 raccoons, 5.7%, 95% confidence interval (CI) 7.8-3.5%; three masked palm civets, 2.0%, 95% CI 4.2-0%]. Many of the isolates belonged to serovars that are commonly isolated from human gastroenteritis patients (e.g. S. Infantis, S. Typhimurium, and S. Thompson). The antimicrobial susceptibility test showed that 26.9 % of the isolates from raccoons were resistant to at least one antimicrobial agent, whereas none of the isolates from masked palm civets were resistant. Yersinia sp. was detected from 193 samples (177 raccoons, 38.6%, 95% CI 43.0-34.1%; 16 masked palm civets, 10.5%, 95% CI 15.3-5.6%). All virulent Yersinia strains belonged to Yersinia pseudotuberculosis, which was isolated from seven (1.5%, 95% CI 2.6-0.4%) raccoons and six (3.9%, 95% CI 7.0-0.8%) masked palm civets. According to the detection of virulence factors, all the Y. pseudotuberculosis isolates belonged to the Far Eastern systemic pathogenicity type. Campylobacter spp. was detected from 17 samples (six raccoons, 1.3%, 95% CI 2.3-0.3%; 11 masked palm civets, 7.2%, 95% CI 11.3-3.1%). Among these, three isolates from raccoons were identified as C. jejuni. These results showed that these pathogens can be transmitted by human activities, other wild animals, and the environment to feral raccoons and masked palm civets, and vice versa. As these animals have omnivorous behaviour and a wide range of habitats, they can play an important role in the transmission of the enteric pathogens.

Pathological changes in captive monkeys with spontaneous yersiniosis due to infection by Yersinia enterocolitica serovar O8.

An outbreak of fatal yersiniosis due to infection with Yersinia enterocolitica serovar O8 is documented in two species of captive monkey. Five of 50 squirrel monkeys (Saimiri sciureus) and one of two agile gibbons (Hylobates agilis) died following several days of diarrhoea. Necropsy examination revealed necrotizing enterocolitis and multifocal necrosis or abscesses in various organs. Microscopically, these lesions comprised multifocal necrosis with bacterial colonies, neutrophils and accumulation of nuclear debris. Occasional lesions included macrophages and abscess formation. Immunohistochemically, the bacteria were identified as Y. enterocolitica O8. In addition, Y. enterocolitica serotype O8 was isolated from animal organs in pure culture. This is the first report of fatal cases of infection with Y. enterocolitica serovar O8 in animals.

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay.

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.