Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010-2011 winter season in Japan.

H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.

Relationship between phylogenetic groups of Escherichia coli and Pathogenicity among Isolates from chickens with Colibacillosis and healthy chickens.

Avian pathogenic Escherichia coli (APEC) is closely related to extraintestinal pathogenic E. coli, which are frequently assigned to specific phylogenetic groups (phylogroups). Therefore, we investigated the association between phylogroups of E. coli isolates and those recovered from commercial broiler and layer chickens with colibacillosis. We used 104 E. coli isolates from chickens with colibacillosis (hereafter referred to as "colibacillosis-related isolates"), 56 E. coli isolates obtained from fecal samples of clinically healthy broiler chickens, and 58 isolates obtained from environmental samples of layer chicken housing facilities where clinically healthy layer chickens were reared (hereafter referred to as "healthy chicken-related isolates"). The prevalence of phylogroup F among colibacillosis-related isolates was significantly (P < 0.05) higher than that among healthy chicken-related isolates, while phylogroups A and B1 were more frequently distributed in healthy chicken-related isolates. Fifty-seven (87%) of 65 colibacillosis-related isolates belonging to phylogroup F were defined as APEC based on the presence of virulence-associated genes according to a previously established criterion. In contrast, none of the healthy chicken-related isolates were defined as APEC. As evidenced by the chicken embryo lethality assay, 87 of the 92 healthy chicken-related isolates tested had embryo lethality rates of <30% and were considered avirulent, whereas 59 of the 104 colibacillosis-related isolates were considered virulent. Nonetheless, among isolates exhibiting embryo lethality rates of <30%, the mean lethality rate of embryos inoculated with colibacillosis-related isolates was significantly higher than that of embryos inoculated with healthy chicken-related isolates. These observations suggest that phylogroup F predicts colibacillosis among E. coli strains with virulence-associated genes.

Prevalence of qnrS-positive Escherichia coli from chicken in Thailand and possible co-selection of isolates with plasmids carrying qnrS and trimethoprim-resistance genes under farm use of trimethoprim.

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 µg/mL and 32 µg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP.

Genetic Characterization of CTX-M-2-Producing Klebsiella pneumoniae and Klebsiella oxytoca Associated With Bovine Mastitis in Japan.

CTX-M-2-producing Klebsiella oxytoca (K. oxytoca) has not received much attention in animal husbandry compared with Klebsiella pneumoniae (K. pneumoniae), a major reservoir of extended-spectrum ß-lactamase (ESBL) genes. Bacteriological examinations of 1,466 mastitic milk samples between October 2012 and December 2014 were conducted. Ninety-five K. pneumoniae isolates (total prevalence: 6.5%) and 81 K. oxytoca isolates (total prevalence: 5.5%) were obtained. Seventeen K. pneumoniae isolates obtained from 15 animals reared on 11 farms and 9 K. oxytoca isolates obtained from 9 animals reared on the same farm were phenotypically confirmed to be ESBL producers. All nine ESBL-producing K. oxytoca isolates were obtained from one farm between June and November 2013 and related to a significantly (p < 0.05) higher monthly prevalence of mild mastitis (in June, August, September, October, and November 2013). Pulsed-field gel electrophoresis (PFGE) patterns of ESBL-producing K. pneumoniae isolates were distinguished from each other by more than 6-band differences except for two isolates from two animals, whereas all nine K. oxytoca isolates showed an identical PFGE pattern. Transferability of the bla CTX-M-2 gene was found in 14 K. pneumoniae and 9 K. oxytoca isolates by conjugation analysis. Of these isolates, the bla CTX-M-2 gene was detected on plasmids belonging to the incompatibility (Inc) groups P and N derived from five K. pneumoniae and nine K. oxytoca isolates, respectively, although the plasmids from the remaining nine K. pneumoniae were untypeable. All the transconjugants exhibited elevated minimum inhibitory concentrations of ampicillin, cefotaxime, and ceftiofur compared with those in the wild-type, recipient strain. Restriction fragment length polymorphism analysis demonstrated that the IncN plasmids extracted from eight of nine transconjugants, which received resistance against ß-lactams from K. oxytoca, showed an identical DraI digestion pattern. These results suggest that the CTX-M-2-producing K. oxytoca strain with the above-mentioned characteristics may have clonally spread within a farm, whereas the bla CTX-M-2 gene in K. pneumoniae possibly disseminated among the farms through different plasmids. Thus, monitoring of ESBL genes, including the bla CTX-M-2 gene, among causative agents of bacterial mastitis in cows can help to develop relevant treatments and control practices.

Susceptibility of two species of wild terrestrial birds to infection with a highly pathogenic avian influenza virus of H5N1 subtype.

The recent epidemic caused by H5N1 highly pathogenic avian influenza (HPAI) viruses has spread over many parts of Asia, Europe and Africa. Wild birds, particularly waterfowl, are considered to play a role in viral dissemination. However, detailed information on whether wild terrestrial birds act as carriers is currently unavailable. To investigate the susceptibility of terrestrial birds to HPAI viruses, two species of wild bird (great reed warbler and pale thrush) that are common in East Asia were infected with H5N1 HPAI virus. The results showed that both species were highly susceptible to the virus. The great reed warbler showed fatal infection with 100% mortality, but the pale thrush survived for longer periods (>8 days) with viral shedding. These findings suggest that there is variation in clinical outcome after infection of wild terrestrial birds, and that some bird species could become subclinical excretors of the H5N1 virus.

Salmonella isolated from the feces of migrating cranes at the Izumi Plain (2002-2008): serotype, antibiotic sensitivity and PFGE type.

From November 2002 to February 2008, 2,251 crane feces were collected at the Izumi Plain in Kagoshima Prefecture. Salmonella enterica was isolated from 359 feces (15.9%), of which 332 (92.5%) were Salmonella Typhimurium (ST), 9 were S. Hvittingfoss/II, 4 were S. Abaetetuba, 3 were S. Enteritidis, 2 were S. Konstanz, 1 was S. Pakistan and 8 were untyped isolates, respectively. Against 12 antimicrobial agents, no resistant strains were found in 154 isolates examined, but one was found to be resistant to ampicillin. By pulsed-field gel electrophoresis (PFGE), all but one of the 68 ST isolates tested showed indistinguishable banding patterns; one had a different pattern. The results suggest that ST strains from the same origin would spread in crane flocks during their stay at Izumi Plain every winter.

Development of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of avian influenza viruses in field specimens.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.

Research Note: Longitudinal monitoring of chicken houses in a commercial layer farm for antimicrobial resistance in Escherichia coli with special reference to plasmid-mediated quinolone resistance.

Plasmid-mediated quinolone resistance (PMQR) genes located on conjugative plasmids can be transferred to other bacteria in the absence of antimicrobial selective pressure. To elucidate the prevalence of resistance, including PMQR in an egg-producing commercial layer farm in western Japan where no antimicrobials were used, minimum inhibitory concentrations (MIC) for a total of 375 Escherichia coli isolates obtained from chicken houses in the farm between 2012 and 2017 were determined using the agar dilution methods. Eighty-seven isolates resistant to oxytetracycline (OTC) accounted for 23.0% of the tested isolates, followed by isolates resistant to dihydrostreptomycin (DSM) (18.4%), sulfisoxazole (18.1%), ampicillin (AMP) (14.4%), trimethoprim (TMP) (14.4%), and nalidixic acid (10.1%). The prevalence rate of multidrug-resistant (MDR) isolates-which are resistant to 3 or more antimicrobial classes, including ß-lactams, aminoglycosides, quinolones, folate pathway inhibitors, tetracyclines, and phenicols-was inversely related to the age of chickens at the time of bacterial examination. Probably, the prevalence of MDR isolates in layer chickens may have decreased with age owing to the absence of selective pressure. Furthermore, 45 isolates exhibiting enrofloxacin MICs of more than 0.25 µg/mL were examined for PMQR genes. The transfer of PMQR genes was tested by conjugation analysis. Southern blot analysis of genomic DNA revealed that the qnrS1 (5 isolates), qnrS2 (1 isolate), and qnrS13 genes (1 isolate) were located on plasmids with sizes ranging from approximately 60 to 120 kpb. In 1 of the 5 qnrS1-positive isolates and in an isolate with qnrS13, the qnrS genes were transferred to recipient strains. The plasmid harboring the qnrS1 gene was typed as IncF by PCR-based replicon typing. On this plasmid, the blaTEM, aadA, tetA, and dfrA1 genes responsible for resistance to AMP, DSM, OTC, and TMP, respectively, were detected. The tetA gene was detected in the plasmid harboring the qnrS13 gene, which was typed as IncI1. These results suggest that despite the low prevalence of quinolone resistance in this farm, various PMQR genes, located on diverse plasmids, exist.

Outbreaks of highly pathogenic avian influenza in zoo birds caused by HA clade 2.3.4.4 H5N6 subtype viruses in Japan in 2016.

In late 2016, two zoos, one in northern Japan and the other in central Japan, experienced highly pathogenic avian influenza (HPAI) outbreaks, in which multiple zoo birds were infected with H5N6 subtype HPAI virus (HPAIV). Here, we report an overview of these HPAI outbreaks. HPAIV infections were confirmed by virus isolation in three black swans (Cygnus atratus) and three snowy owls (Bubo scandiacus) kept in the Omoriyama Zoo hospital. At Higashiyama Zoo and Botanical Gardens, following the death of a black swan at a zoo pond, nine waterfowl, including two black swans, four cackling geese (Branta hutchinsii leucopareia), two mallards (Anas platyrhynchos), and a wigeon (Anas penelope), died after HPAIV infection in isolation facilities. Based on the presence of H5-specific antibodies in their sera, two surviving black swans and a surviving mallard at Higashiyama Zoo appeared to have HPAIV infection, although the virus was not isolated. The detectable levels of antibodies (≥10 HI) were maintained for at least 5-9 months, as determined by haemagglutinin inhibition test. Isolation of two H5N6 subtype HPAIVs from an open-air pond where affected zoo birds were previously housed at Higashiyama Zoo strongly indicates that wild waterfowl associated with aquatic environments brought the virus to the zoo. The phylogenetic relationships of the 18 isolates indicated direct viral transmission among birds within each zoo. In both zoos, containment of suspected birds in isolation facilities might have allowed the virus spread among birds inside the facility. However, maintaining containment measures and strict sanitation procedures could facilitate successful physical containment and clearance of HPAIV in both zoos.

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008.

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007-2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.

Multiple routes of entry for Escherichia coli causing colibacillosis in commercial layer chickens.

Colibacillosis associated with salpingitis occurred in a layer chicken flock on a commercial egg-producing farm in Japan. An increase in mortality was observed when the birds were at 62 weeks of age and reached 0.89% at 68 weeks of age. Postmortem examinations revealed pericarditis, perihepatitis, airsacculitis, and reproductive tract lesions in 4 affected birds at 69 weeks of age. Analysis of pulse-field gel electrophoresis (PFGE) patterns and putative virulence genes of 22 E. coli isolates obtained from the affected birds demonstrated that isolates from liver, heart, and the surface of the reproductive tract of one bird were genetically unrelated with those recovered from the lumen of the oviduct. In the other birds, isolates from liver, heart, and reproductive tract lesions were closely related to each other. These findings suggest that salpingitis in the former bird may be caused by ascending infection of the oviduct from the cloaca and salpingitis in the remaining birds may occur as part of systemic infection.

Plasmid-mediated quinolone resistance in Escherichia coli isolates from commercial broiler chickens and selection of fluoroquinolone-resistant mutants.

Plasmid-mediated quinolone resistance (PMQR) is a potential concern for animal husbandry and public health. Escherichia coli isolates from a total of 109 fecal samples collected from 6 commercial broiler farms between 2007 and 2011 were examined for PMQR genes, and transfer of these genes was tested by conjugation analysis to elucidate the prevalence and spread of PMQR in broiler chickens. Two isolates from 2 farms harbored the aac(6')-Ib-cr gene that was not detected in plasmids using Southern blot analysis of S1 nuclease-digested genomic DNA separated by pulsed-field gel electrophoresis. In these 2 isolates, nucleotide mutations in the gyrA and parC genes that result in amino acid substitutions were detected. Additionally, a total of 6 isolates originating from 6 chickens from the 2 farms were positive for the qnrS1 gene. In 2 of the 6 isolates, the qnrS1 gene was transferred to a recipient strain. Two transconjugants harboring the qnrS1 gene were cultured on media supplemented with successively higher concentrations of enrofloxacin (ERFX). After a 5-time subcultivation, the ERFX MICs reached 8 and 16 µg/mL, and no nucleotide mutations were detected in the gyrA, gyrB, parC, and parE genes. Our results suggest that the prevalence of PMQR was relatively low in broiler chickens and that exposure of bacteria carrying PMQR genes to the selective pressure of fluoroquinolones can result in resistance to fluoroquinolone, which is not caused by mutations in genes encoding topoisomerases.

Isolation of Campylobacter jejuni and coliform bacilli from bile and liver obtained from slaughter cattle in Western Japan.

A total of 290 bile samples from 143 Japanese Brown, 97 Japanese Black, and 50 Holstein cattle, and a total of 148 liver samples from 81 Japanese Brown, 49 Japanese Black, and 18 Holstein cattle were examined for the presence of Campylobacter jejuni by direct plating. The bile samples were also subjected to enumeration of coliform bacilli. Sixty-eight (23%) bile samples and 2 (1.4%) liver samples were positive for C. jejuni. A significantly higher isolation rate was observed from bile samples from Holstein (50%) than from Japanese Black (22%) and Japanese Brown (15%) cattle. C. jejuni was isolated from 52 of 232 bile samples that contained < 30 CFU/ml (under the detection threshold) of coliform bacilli. The presence of C. jejuni from bile was observed throughout the year. Fifty-four of the 68 bile isolates were serologically typed into eight groups. Serotypes O:4 complex (28 isolates) and O:2 (11 isolates), which were commonly isolated from human patients in Japan, accounted for 57% of the isolates. These observations suggest that bile can be a cause of contamination with C. jejuni even though it contains only a low number of coliforms.

Genotypic and phenotypic characterization of Salmonella enterica subsp. enterica serovar Typhimurium monophasic variants isolated in Thailand and Japan.

Monophasic variants of Salmonella enterica serovar Typhimurium isolated in Thailand and Japan were characterized to elucidate the genetic basis of the monophasic phenotype, genetic relatedness, and antimicrobial resistance. A total of 20 Salmonella isolates agglutinated with anti-O4 and anti-H:i serum and not agglutinated with either anti-H:1 or anti-H:2 serum were identified as monophasic variants of Salmonella serovar Typhimurium because they harbored IS200, specific to this serovar, and lacked the fljB gene. An allele-specific PCR-based genotyping method that detects a clade-specific single nucleotide polymorphism indicated that seven swine isolates and one human isolate from Thailand were grouped into clade 1; five isolates from layer chicken houses and layer chicken feces from Japan were grouped into clade 8, together with two Salmonella serovar Typhimurium isolates from chicken houses in Japan; and five isolates from swine feces from Thailand and two isolates from layer chicken feces from Japan were grouped into clade 9. Multilocus sequencing typing demonstrated that sequence type (ST) 34 isolates were solely grouped into clade 9. Clade 1 and 8 isolates were assigned as ST19. Pulsed-field gel electrophoresis revealed multiple types within each of the clades. The presence of antimicrobial resistance genes and plasmid replicon type, of the clade 1 and 9 isolates were comparable to those reported for epidemic strains of monophasic variants. Our results suggest that monitoring monophasic variants of serovar Typhimurium is important for understanding of the spread of these variants in Thailand and Japan.

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm.

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.

Aspiration pneumonia as a cause of neonatal death in three captive bottlenose dolphins (Tursiops truncates).

Neonatal weakness of calves is one of the common reproductive-related problems in captive cetaceans; clinical signs can be observed in the first few hours after delivery. Three 3-day-old bottlenose dolphins died with history of weakness since birth. Pathological study demonstrated purulent bronchopneumonia associated with prominent bacterial colonies and foreign substances in alveoli, suggesting aspiration pneumonia as a cause of neonatal weakness and resultant death of the three calves.

Screening of the enterocin genes and antimicrobial activity against pathogenic bacteria in Enterococcus strains obtained from different origins.

Antimicrobial activities of 139 Enterococcus isolates (48 E. faecium and 91 E. faecalis) obtained from canine feces, boiler meat samples, swine feces, wild waterfowl feces, and human feces were examined against respective bacteria, including Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli. Bacteriocin (BAC) production assay revealed that the antimicrobial activity against at least one of 6 indicator strains (BAC+ phenotype) was found in 51 (37%) isolates (29 E. faecium and 22 E. faecalis). Twenty-four of 46 isolates positive for at least one of the enterocin structural genes (entA, entB, entL50AB, and cylL) showed a BAC+ phenotype. The existence of other enterocins or nonenterocin factors was implied because the BAC+ phenotype was detected in a total of 27 Enterococcus isolates that had none of the enterocin genes tested. The antimicrobial activity against Gram-negative strains (Salmonella Enteritidis and E. coli) was detected in the 6 Enterococcus isolates that had either the entA, entB, entL50AB or cylL genes. Moreover, the proportion of the antimicrobial activity against L. monocytogenes among the cylL-positive E. faecalis isolates showing beta-hemolysis (10/16) was significantly (p<0.01) higher than among those lacking beta-hemolysis (2/15). The results suggested that certain characteristics are likely to be associated with the antimicrobial activity against specific organisms.

Mitral stenosis with bacterial myocarditis in a cat.

An eleven-year-old female Japanese mongrel cat was referred to the Tottori University Veterinary Teaching Hospital for assessment of acute paresis and dyspnea. Two-dimensional echocardiography showed a hydropericardium. The mitral valve leaflets were thickened, the separation of the right and left leaflets was not complete. Treatments with intravenous fluids of lactate Ringer solution, furosemide, urokinase, antibiotics were initiated, but did not improve the respiratory failure. The cat died 10 days later. From pathological and microbiological examinations, this was an unusual case diagnosed as acquired mitral stenosis associated with congenital malformation of the mitral valve complex, and accompanied by secondary infectious myocarditis with Streptococcus canis.

Strong antiviral activity of heated and hydrated dolomite--preliminary investigation.

Heated and hydrated naturally occurring dolomite showed very strong antiviral activity. Infectivity of avian and human influenza, avian infectious bronchitis (coronavirus), Newcastle disease (paramyxovirus) and avian laryngotracheitis (herpesvirus) viruses dropped at least 1,000 fold following contact with the dolomite for five minutes at 4 degrees C. Dolomite is expected to be useful to inhibit the incidence of emerging and re-emerging infectious diseases.

Inhibitory effects of Enterococcus strains obtained from a probiotic product on in vitro growth of Salmonella enterica serovar enteritidis strain IFO3313.

Enterococcus faecium and Enterococcus gallinarum strains were isolated from a commercial probiotic product and the effects of these strains on the growth of Salmonella enterica serovar Enteritidis strain IFO3313 were investigated. Viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. faecium were significantly (P < 0.05) lower than those in pure cultures after 6, 8, and 24 h when the cultures were incubated in heart infusion broth at 37 and 41 degrees C. Significant differences in viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. gallinarum and those in pure cultures were also observed after 8 and 24 h at 37 and 41degrees C. Similar observations were shown in mixed cultures of Salmonella Enteritidis with the reference strains of E. faecium GIFU8355 and E. gallinarum ATCC 49573. Significant differences in viable cell counts of these enterococcal strains were not shown among pure and mixed cultures with Salmonella Enteritidis. The pH values in pure and mixed cultures were 7.0 or 7.5 throughout the experiments. E. faecium strains were found to harbor the genes encoding enterocins A and B and showed inhibitory zones with a diameter of 4 to 6 mm against growth of Salmonella Enteritidis in the enterocin production assays. However, the E. gallinarum strains possessed neither of the enterocin genes tested and exhibited no inhibition zone in the enterocin production assays. These results indicated that enterococcal strains exhibit inhibitory effects on the growth of Salmonella Enteritidis and these effects were due to both enterocin and nonenterocin factors.