Monocytogenes Serotype 4b Research Articles

Effect of acidic conditions on the growth and expression of two virulence genes of Listeria monocytogenes serotype 4b

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA, of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA-based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments.

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence, resistance to biocides, genomic landscape and biocontrol.

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.

Control of Listeria monocytogenes growth and virulence in a traditional soft cheese model system based on lactic acid bacteria and a whey protein hydrolysate with antimicrobial activity

“Torta del Casar” is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a “Torta del Casar” cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in “Torta del Casar” cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures.

Transcriptomic Response of L. monocytogenes to Co-Culture with S. cerevisiae

The aim of the present study was to assess the transcriptomic response of L. monocytogenes during co-culture with three S. cerevisiae strains. For this purpose, BHI broth was inoculated with 7 log CFU·mL−1 L. monocytogenes serotype 4b strain LQC 15257, isolated from a strawberry sample and 4 log CFU·mL−1 S. cerevisiae strains Y32, Y34 and Y37, isolated from spontaneous olive fermentation. Sampling took place after 24 and 48 h incubation at 5 and 20 °C. RNA was extracted, stabilized and the transcription of virulence associated genes prfA, sigB, hly, plcA, plcB, inlA, inlB, inlC and inlJ, was assessed by RT-qPCR. Co-culture with the yeast strains mostly affected the transcription of sigB and inlJ, the upregulation of which during growth at 5 °C for 24 h, reached 10.13 and 9.76 log2(fold change), respectively. Similarly, the effect that incubation time had on the relative transcription of the genes under study was dependent on the co-cultivating yeast strain. On the other hand, the effect of the yeast strain was less pronounced when the relative transcription of the genes under study was assessed between 20 °C and 5 °C. In that case, incubation temperature seemed to have an important effect since, in the 79.2% of the samples analyzed, upregulation was evident, irrespective of yeast strain presence. These results highlight the complex trophic relationships that take place during co-existence between L. monocytogenes and S. cerevisiae.

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.

Identification and Characterization of a Novel Genomic Island Harboring Cadmium and Arsenic Resistance Genes in Listeria welshimeri.

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2′s cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 μg/mL) than other cadAC cassettes (MIC ≥ 140 μg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.

Listeriosis Caused by Persistence of Listeria monocytogenes Serotype 4b Sequence Type 6 in Cheese Production Environment

Abstract A nationwide outbreak of human listeriosis in Switzerland was traced to persisting environmental contamination of a cheese dairy with Listeria monocytogenes serotype 4b, sequence type 6, cluster type 7488. Whole-genome sequencing was used to match clinical isolates to a cheese sample and to samples from numerous sites within the production environment.

Listeriosis Caused by Persistence of Listeria monocytogenes Serotype 4b Sequence Type 6 in Cheese Production Environment.

A nationwide outbreak of human listeriosis in Switzerland was traced to persisting environmental contamination of a cheese dairy with Listeria monocytogenes serotype 4b, sequence type 6, cluster type 7488. Whole-genome sequencing was used to match clinical isolates to a cheese sample and to samples from numerous sites within the production environment.

Comprehensive proteomic analysis and pathogenic role of membrane vesicles of Listeria monocytogenes serotype 4b reveals proteins associated with virulence and their possible interaction with host

Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection.

Prevalence and diversity of Salmonella spp., Campylobacter spp., and Listeria monocytogenes in two free-range pig slaughterhouses

Salmonella spp., Campylobacter spp. and Listeria monocytogenes have a significant impact on public health with slaughterhouses providing many opportunities for the proliferation of pathogenic bacteria. To evaluate the prevalence and diversity of these microorganisms along the free-range pork production chain, a total of 750 samples (5 samples/animal; 15 animals/farm; 5 farms/slaughterhouse) were collected from two slaughterhouses and analysed by specific ISO methodologies. Salmonella spp. (12.93%, CI95 10.72–15.52%), Campylobacter spp. (17.17%, CI95 13.00–21.74%) and L. monocytogenes (9.07%, CI95 7.21–11.33%) were recovered at different stages of the production chain, with the highest prevalence detected in tonsils for Salmonella spp. (30.67%, CI95 23.85–38.44%) and L. monocytogenes (39.33%, CI95 31.87–47.32%) and in faeces for Campylobacter spp. (57.33%, CI95 49.33–64.96%). Thirteen different Salmonella serotypes were detected with monophasic Salmonella Typhimurium as the most frequent one. C. coli, C. jejuni and L. monocytogenes serotype 4b and 1/2a were also identified. A significant higher prevalence of Salmonella spp. in total and from skin samples in slaughterhouse B than in slaughterhouse A was detected. In addition, a higher, although not significant, prevalence of the selected pathogens was observed in meat samples from slaughterhouse B with respect to slaughterhouse A (10.67% vs 0% for Campylobacter spp.; and 4% vs 0% for Salmonella spp. and L. monocytogenes). Our results highlight the risk of contamination of pork meat by the microorganisms under study and point out the importance of implementing specific control measures.

Comparison of oxidative stress response and biofilm formation of Listeria monocytogenes serotypes 4b and 1/2a

Among 13 serotypes of Listeria monocytogenes, serotypes 4b and 1/2a account for most of the sporadic listeriosis cases and environmental isolations. To understand the mechanism of stress response in these serotypes, six strains of each serotype were selected in this study to differentiate the physiological and genetic response of these L. monocytogenes under oxidative stress. Oxidative stress tolerance was examined by plate count after hydrogen peroxide exposure during exponential phase, long-term-survival phase and biofilm state. The biofilm forming capability of the two serotypes was also compared using crystal violet assay. Three oxidation resistance genes (kat, sod and fri), two stress-regulator encoding genes (sigB and perR), and one DNA repair gene (recA) were selected for transcription analysis by quantitative RT-PCR. Most serotype 4b strains exhibited a stronger resistance to 0.6% H2O2 than serotype 1/2a during exponential phase and long-term-survival phase, while the latter demonstrated stronger biofilm forming capacity in BHI broth at 37 °C. At the genetic level, there were significant differences in the expression of three genes (sod, fri and perR) under oxidative stress between two serotypes, which suggests a role in the observed variation towards stress resistance. In addition, the expression of recA was induced under oxidative stress in L. monocytogenes strains of both serotypes.

A Comprehensive Evaluation of the Genetic Relatedness of Listeria monocytogenes Serotype 4b Variant Strains.

Recently, we have identified a link between four listeriosis incidents/outbreaks to a variant of Listeria monocytogenes (Lm) serotype 4b strains, 4bV. Although 4bV strains have been reported from clinical specimens as well as from foods, listeriosis outbreaks occurring in 2014–2016 were the first reported outbreaks involving 4bV in the USA. Since traditional typing methods do not detect members of this group, we undertook a systematic and retrospective analysis of all Lm in the NCBI WGS Sequence Read Archive database to investigate the burden of 4bV strains among all listeriosis cases. This analysis identified the presence of isolates causing sporadic cases as well as those associated with the aforementioned outbreaks, as determined by WGS and traditional epidemiology. In total, approximately 350 Lm 4bV strains were identified from multiple parts of the USA as well as from Australia and Chile, dating back to 2001. The genomic relatedness of these strains was compared using the CFSAN SNP Pipeline and multi-virulence-locus sequence typing (MVLST). Using the CFSAN Pipeline tool, the 4bV strains were found to group into seven clusters that were separate from 4b strains. All seven clades appeared to contain isolates from both clinical and non-clinical sources. Conversely, the MVLST analysis revealed that practically all of the strains belonged to a single clade, suggesting that 4bV strains from disparate geographic regions and sources are under varied selective pressure, restricting diversity across these six virulence loci while allowing more variability across the genome as a whole. Further evaluation of these 4bV strains identified genes potentially acquired from a lineage II source external to the lmo0733–lmo0739 region, as well as highly conserved SNPs unique to the 4bV strains when compared to those from other lineages. Taken together, these data suggest that 4bV strains have undergone adaptive responses to selective pressures that may enhance survival in the environment while maintaining the pathogenic potential of serotype 4b strains.

A clade of Listeria monocytogenes serotype 4b variant strains linked to recent listeriosis outbreaks associated with produce from a defined geographic region in the US

Four listeriosis incidences/outbreaks, spanning 19 months, have been linked to Listeria monocytogenes serotype 4b variant (4bV) strains. Three of these incidents can be linked to a defined geographical region, while the fourth is likely to be linked. In this study, whole genome sequencing (WGS) of strains from these incidents was used for genomic comparisons using two approached. The first was JSpecies tetramer, which analyzed tetranucleotide frequency to assess relatedness. The second, the CFSAN SNP Pipeline, was used to perform WGS SNP analyses against three different reference genomes to evaluate relatedness by SNP distances. In each case, unrelated strains were included as controls. The analyses showed that strains from these incidents form a highly related clade with SNP differences of ≤101 within the clade and >9000 against other strains. Multi-Virulence-Locus Sequence Typing, a third standardized approach for evaluation relatedness, was used to assess the genetic drift in six conserved, known virulence loci and showed a different clustering pattern indicating possible differences in selection pressure experienced by these genes. These data suggest a high degree of relatedness among these 4bV strains linked to a defined geographic region and also highlight the possibility of alterations related to adaptation and virulence.

Local Outbreak of Listeria monocytogenes Serotype 4b Sequence Type 6 Due to Contaminated Meat Pâté.

In January and February 2016, five cases of confirmed and two cases of probable infection due to Listeria monocytogenes serotype 4b, sequence type (ST) 6 belonging to a single pulsed-field gel electrophoresis pulsotype pattern were registered in a region of southern Switzerland. L. monocytogenes was detected in blood samples (four cases) and pleural fluid (one case). Furthermore, L. monocytogenes 4b ST6 was detected in a stool sample of an asymptomatic person exposed to a common food. Forthwith, the food safety authority and a local gourmet meat producer reported L. monocytogenes contamination of meat pâté. Analysis of further food and environmental samples from the premises of the producer yielded isolates matching the clinical strains and confirmed the presence of L. monocytogenes 4b ST6 in the mincing machine as the cause of the food contamination.

Isolation, characterization and evaluation of virulent bacteriophages against Listeria monocytogenes

Bacteriophages are considered a potential antimicrobial food intervention due to their ability to inactivate target bacteria in different environments. This study was undertaken to isolate, characterize, and identify a mixture of phages that could be used for control of Listeria monocytogenes. A total of 37 phages were isolated from raw sewage sludge using L. monocytogenes serotypes 4b, 1/2a and 1/2b strains. The host range of these phages was determined by spot-testing. Ten lytic phages were selected for further evaluation with efficiency of plating (EOP) analysis and characterization by pulsed field gel electrophoresis (PFGE). According to the spot tests and EOP results, the isolated phages against L. monocytogenes 4b strains were found to be capable of lysing a wider range of ribotypes. Also, three phages specific for 1/2a and 1/2b serotypes had similar lysis capabilities over a range of target Listeria with high EOP values between 0.5 and 1.0. PFGE analysis identified 5 distinct restriction profiles of the 10 selected phages whose DNA was digested with either EcoRI or HinDIII. Transmission electron microscopy (TEM) pictures revealed that five selected phages belonged to the Myoviridae and one phage to the Podoviridae family. Based on screening results, a phage cocktail (LP1) comprised of six lytic phages was used to inactivate a mixed culture of five L. monocytogenes strains. LP1 was able to lyse bacterial cells within 30–60 min at multiplicity of infection values (MOI, PFU/CFU) of 1000 and maintained the target cells under detection levels for 24 to 48 h at 4, 10, and 30 °C. These results suggested the LP1 phage collection contained effective and strongly lytic phages that may serve as antibacterial preparation to control L. monocytogenes of different serotypes.

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools.

The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. L. monocytogenes is traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface of L. monocytogenes strains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolate L. monocytogenes from food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of the L. monocytogenes serotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range of L. monocytogenes isolates.

Draft Genome Sequences of Listeria monocytogenes Serotype 4b Strains 944 and 2993 and Serotype 1/2c Strains 198 and 2932.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis among humans and animals. The draft genome sequences of L. monocytogenes serotype 4b strains 944 and 2993 and serotype 1/2c strains 198 and 2932 are reported here.

Molecular characterization of Listeria monocytogenes in bovine milk and evaluating the sensitivity of PCR for direct detection in milk

Food-borne listeriosis, recognized as an emerging bacterial disease of humans and animals worldwide, is caused by L. monocytogenes with at least 95% of the strains isolated from foods and patients belonging to serovars 1/2a, 1/ 2b and 4b. Milk and dairy products were implicated as sources of listeriae in several widely publicized incidents, thus suggesting that the mammary glands of mastitic cattle may be an important reservoir of Listeria. In the present study, 350 bovine milk samples were collected for prevalence and molecular characterization studies of Listeria spp. The isolates were phenotypically and genotypically characterized by biochemical tests, haemolysis on sheep blood agar, CAMP test, PI-PLC assay and multiplex PCR targeting virulence cluster genes namely haemolysin (hlyA), PI-PLC (plcA), actin (actA), p60 (iap) and regulatory (prfA); along with multiplex PCR for typing major serovars targeting lmo0737, ORF2819, ORF2110 and prs genes. Four pathogenic L. monocytogenes were recovered indicating prevalence rate of 1.14% in milk while the overall prevalence rate of Listeria spp. was 1.42%. All the four pathogenic isolates were characterized as L. monocytogenes serotype 4b. Antibiogram of the pathogenic L. monocytogenes isolates revealed sensitivity for amikacin, gentamycin, norfloxacin and doxycyclin. Animal sera (169) screened by indirect ELISA for antibodies against listeriolysin O showed sero-positivity of 7.1%. Sensitivity of PCR for direct detection from milk was evaluated to be 8.8 × 105 L. monocytogenes cells/ml of milk. Thus, the presence of pathogenic strains of L. monocytogenes in raw milk appeared to be a cause for concern with profound public health implications.

Fresh Produce–Associated Listeriosis Outbreaks, Sources of Concern, Teachable Moments, and Insights

Foodborne transmission of Listeria monocytogenes was first demonstrated through the investigation of the 1981 Maritime Provinces outbreak involving coleslaw. In the following two decades, most listeriosis outbreaks involved foods of animal origin, e.g., deli meats, hot dogs, and soft cheeses. L. monocytogenes serotype 4b, especially epidemic clones I, II, and Ia, were frequently implicated in these outbreaks. However, since 2008 several outbreaks have been linked to diverse types of fresh produce: sprouts, celery, cantaloupe, stone fruit, and apples. The 2011 cantaloupe-associated outbreak was one of the deadliest foodborne outbreaks in recent U.S. history. This review discusses produce-related outbreaks of listeriosis with a focus on special trends, unusual findings, and potential paradigm shifts. With the exception of sprouts, implicated produce types were novel, and outbreaks were one-time events. Several involved serotype 1/2a, and in the 2011 cantaloupe-associated outbreak, serotype 1/2b was for the first time conclusively linked to a common-source outbreak of invasive listeriosis. Also in this outbreak, for the first time multiple strains were implicated in a common-source outbreak. In 2014, deployment of whole genome sequencing as part of outbreak investigation validated this technique as a pivotal tool for outbreak detection and speedy resolution. In spite of the unusual attributes of produce-related outbreaks, in all but one of the investigated cases (the possible exception being the coleslaw outbreak) contamination was traced to the same sources as those for outbreaks associated with other vehicles (e.g., deli meats), i.e., the processing environment and equipment. The public health impact of farm-level contamination remains uncharacterized. This review highlights knowledge gaps regarding virulence and other potentially unique attributes of produce outbreak strains, the potential for novel fresh produce items to become unexpectedly implicated in outbreaks, and the key role of good control strategies in the processing environment.

Outbreak of Listeria Monocytogenes in Pheasants

Listeria monocytogenes is capable of infecting almost all animals. However, outbreaks of listeriosis are infrequent in birds. This report describes an outbreak of listeriosis in a small pheasant (Phasianus colchicus) breeder farm with more than 2,000 pheasants from Hubei province of the People's Republic of China. The affected flock consisted of adult and young birds. Approximately 300 young birds and a few adult birds were found dead within a few days of the onset of clinical signs. Twenty-five dead birds were collected for further examination. Histopathological lesions in the visceral organs were characterized by monocyte infiltration and proliferation. Localized encephalitis and meningitis were detected in the brains of dead birds. Gram-positive organisms were observed in heart blood smear, liver, and brain impression smears. The organisms were isolated from fresh liver and were identified as L. monocytogenes serotype 4b based on multiplex polymerase chain reaction (PCR) and hlyA gene sequence analysis. This is the first report describing outbreak of listeriosis in pheasant flock.