Human Listeriosis.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that can cause severe invasive infections upon ingestion with contaminated food. Clinically, listerial disease, or listeriosis, most often presents as bacteremia, meningitis or meningoencephalitis, and pregnancy-associated infections manifesting as miscarriage or neonatal sepsis. Invasive listeriosis is life-threatening and a main cause of foodborne illness leading to hospital admissions in Western countries. Sources of contamination can be identified through international surveillance systems for foodborne bacteria and strains' genetic data sharing. Large-scale whole genome studies have increased our knowledge on the diversity and evolution of L. monocytogenes, while recent pathophysiological investigations have improved our mechanistic understanding of listeriosis. In this article, we present an overview of human listeriosis with particular focus on relevant features of the causative bacterium, epidemiology, risk groups, pathogenesis, clinical manifestations, and treatment and prevention.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Outbreak of Listeriosis Likely Associated with Baker's Yeast Products, Switzerland, 2022-2024.

We traced back a nationwide outbreak of human listeriosis in Switzerland to a persisting production line contamination of a factory producing baker's yeast with Listeria monocytogenes serotype 1/2a sequence type 3141. We used whole-genome sequencing to match clinical isolates to isolates from product samples.

Clinical and microbiological characteristics and follow-up of invasive Listeria monocytogenes infection among hospitalized patients: real-world experience of 16 years from Hungary.

PURPOSE: Invasive Listeria monocytogenes infection is rare, but can lead to life-threatening complications among high-risk patients. Our aim was to assess characteristics and follow-up of adults hospitalized with invasive L. monocytogenes infection. METHODS: A retrospective observational cohort study was conducted at a national referral center between 2004 and 2019. Patients with proven invasive listeriosis, defined by the European Centre for Disease Prevention and Control criteria, were included. Data collection and follow-up were performed using the hospital electronic system, up until the last documented visit. The primary outcome was in-hospital all-cause mortality, secondary outcomes included residual neurological symptoms, brain abscess occurrence, and requirement for intensive care unit (ICU) admission. RESULTS: Altogether, 63 cases were identified (57.1% male, median age 58.8 ± 21.7 years), and 28/63 developed a complicated disease course (44.4%). At diagnosis, 38/63 (60.3%) presented with sepsis, 54/63 (85.7%) had central nervous system involvement, while 9/63 (14.3%) presented with isolated bacteremia. Frequent clinical symptoms included fever (53/63, 84.1%), altered mental state (49/63, 77.8%), with immunocompromised conditions apparent in 56/63 (88.9%). L. monocytogenes was isolated from blood (37/54, 68.5%) and cerebrospinal fluid (48/55, 87.3%), showing in vitro full susceptibility to ampicillin and meropenem (100% each), gentamicin (86.0%) and trimethoprim/sulfamethoxazole (97.7%). In-hospital all-cause mortality was 17/63 (27.0%), and ICU admission was required in 28/63 (44.4%). At discharge, residual neurological deficits (11/46, 23.9%) and brain abscess formation (6/46, 13.0%) were common. CONCLUSION: Among hospitalized adult patients with comorbidities, invasive L. monocytogenes infections are associated with high mortality and neurological complications during follow-up.

Sodium and its manifold impact on our immune system.

The Western diet is rich in salt, and a high salt diet (HSD) is suspected to be a risk factor for cardiovascular diseases. It is now widely accepted that an experimental HSD can stimulate components of the immune system, potentially exacerbating certain autoimmune diseases, or alternatively, improving defenses against certain infections, such as cutaneous leishmaniasis. However, recent findings show that an experimental HSD may also aggravate other infections (e.g., pyelonephritis or systemic listeriosis). Here, we discuss the modulatory effects of a HSD on the microbiota, metabolic signaling, hormonal responses, local sodium concentrations, and their effects on various immune cell types in different tissues. We describe how these factors are integrated, resulting either in immune stimulation or suppression in various tissues and disease settings.

Gentamicin combination treatment is associated with lower mortality in patients with invasive listeriosis: a retrospective analysis.

PURPOSE: Listeria monocytogenes causes severe bacterial infections with the highest mortality rate among foodborne pathogens in Europe. Combination treatment with ampicillin and gentamicin is recommended for invasive manifestations. However, evidence to support this treatment approach remains limited due to a lack of randomised controlled trials. To explore this critical issue further, we conducted this retrospective, single-center study. METHODS: We identified all patients hospitalized with invasive listeriosis at the University Medical Center Hamburg-Eppendorf between 2009 and 2020 and analyzed the effect of gentamicin combination treatment versus monotherapy on 90-day mortality. RESULTS: In total, 36 patients with invasive listeriosis were included, of which 21 patients received gentamicin combination treatment and 15 received monotherapy. The mean age-adjusted Charlson Comorbidity Index (aaCCI) value was lower in the gentamicin combination treatment group (5.4 vs. 7.4). Neurolisteriosis was more common in the gentamicin group (81% vs. 20%). The 90-day mortality was with significantly lower in the gentamicin combination treatment group (10%) compared to the monotherapy group (60%). Multivariable cox regression analysis, adjusted for a propensity score computed based on neurolisteriosis, aaCCI and sex, revealed a significantly reduced hazard ratio of 0.07 (95% CI: 0.01-0.53, p = 0.01) for 90-day mortality for the gentamicin combination treatment. CONCLUSION: This retrospective study highlights the benefit of gentamicin combination treatment in reducing the 90-day mortality rate among patients with invasive listeriosis. The high prevalence of monotherapy in this study cohort raises concerns about the adequacy of antibiotic therapy in clinical practice.

Recurrent, ICD-associated L. monocytogenes bacteraemia with multiple septic pulmonary embolisms over a 2-year period.

BACKGROUND: Listeria monocytogenes is a bacterial pathogen known for causing listeriosis, a foodborne illness with a wide spectrum of clinical presentations ranging from mild gastroenteritis to severe invasive disease, particularly affecting immunocompromised individuals, pregnant women, newborns, and the elderly. Successful treatment of patients with recurring listeria episodes due to colonised foreign material is often challenging, typically requiring a combination of antimicrobial treatment and surgical removal. CASE PRESENTATION: Here, we present a particularly complex case of chronic invasive listeriosis with a total of six relapses. After extensive investigations, the patient's ICD device was identified as the focus of infection. CONCLUSION: The confirmation of relapses through cgMLST analysis highlights the persistence of Listeria monocytogenes and the potential for recurrence even after apparent resolution of symptoms in patients with foreign material. It emphasises the necessity for a comprehensive assessment to identify and mitigate the risk of relapses, thereby ensuring optimal management and outcomes.

Listeria brain abscess: a therapeutically challenging rare presentation of listeriosis.

We report a very rare case of Listeria multiple brain abscesses manifested as delirium, which represented diagnostic and therapeutic challenges overcome only by the close cooperation between Infectious Diseases and Neuroradiology, without which a satisfactory outcome would not be achieved.An elderly man presented with confusion and drowsiness with a background of type-II diabetes mellitus. Although computed tomography of the brain only showed frontal lobe oedema, contrast magnetic resonance (MR) imaging showed numerous irregular rim-enhancing lesions containing central diffusion restriction, suggesting multiple pyogenic cerebral abscesses of unclear aetiology. Thereafter, Listeria monocytogenes was isolated from blood cultures, suggesting this as the causative organism. Deemed unsuitable for neurosurgical drainage, the patient received medical management with a protracted course of antibiotics. This case was extremely challenging, due to 1) the impossibility of source control, 2) the small number of effective antibiotics available to treat this condition, and 3) the inevitable antibiotic side-effects, derived from long-term exposure. A successful outcome was only possible thanks to strict close multidisciplinary follow up, requiring frequent MR imaging and a judicious antibiotic choice, including monitoring of their side-effects. Due to the rarity of this condition, there is lack of guidance on its management, hence the importance of multidisciplinary involvement with very close imaging and antibiotic monitoring.

Computed tomography and magnetic resonance imaging findings in central nervous system listeriosis.

PURPOSE: To describe the imaging findings and determine the incidence of a characteristic worm-like pattern along the white matter tracts in neurolisteriosis on CT/MRI. METHODS: An IRB-approved retrospective study in 21 consecutive neurolisteriosis cases during January 2002-July 2020. At least one of the following is required: (1) Positive Listeria monocytogenes (LM) in blood with clinical signs of meningeal irritation and/or abnormal CSF profile, (2) positive LM in blood with signs of encephalitis, (3) positive LM in CSF, (4) positive LM from brain biopsy/aspiration. Six cases were excluded due to the lack of contrast-enhanced images, leaving a total of 15 cases for analysis (mean age 53.5 years ± 18.8 SD). The imaging studies were independently reviewed by two blinded readers. Demographic data, imaging findings, and incidence of the worm-like pattern were reported. The Cohen's kappa was used to calculate interrater reproducibility. RESULTS: Of the 12 patients with relevant imaging findings, nine cases (75%) had parenchymal lesions (eight cases in supratentorial compartment and one case in infratentorial compartment), four cases (33.3%) had leptomeningeal enhancement and two cases (16.7%) had hydrocephalus. Brain abscesses were found in eight cases and nodules evocative of abscess in one case. Restricted diffusion in the central area and hemosiderin deposition were observed in all cases. The involvement of white matter tract in a worm-like pattern was demonstrated in eight of nine patients with parenchymal lesions (88.9%). CONCLUSION: Abnormal findings in brain CT/MRI images are common in neurolisteriosis. The incidence of worm-like spread along the white matter tracts is high and may help diagnose suspicious patients.

Unlocking food safety: a comprehensive review of South Africa's food control and safety landscape from an environmental health perspective.

Food fraud (often called fake food in South Africa) the deliberate misrepresentation or adulteration of food products for financial gain, is a growing problem in South Africa (SA) with severe public health and financial consequences for consumers and businesses. The recent public outcry against food fraud practices especially in communities that have lost loved ones due to the consumption of allegedly adulterated foodstuffs, highlights the grave danger that food fraud poses to consumers and the potential for significant reputational damage to food manufacturers. Despite the risks, food fraud often goes undetected, as perpetrators are becoming increasingly sophisticated. The precise magnitude of food fraud remains obscure, as incidents that do not cause consumer illnesses are frequently unreported and, as a result, are not investigated. Food fraud costs the global economy billion annually. This cost is borne by consumers, businesses, and the government. Food fraud can occur at any stage of the food supply chain, from production to processing to retailing or distribution. This is due in part to the limitations of current analytical methods, which are not always able to detect food fraud. This review of food fraud in SA looks at several factors that may be contributing to epidemic of food fraud, including inadequate penalties, inadequate government commitment, a complex labelling regulation, emerging threats such as e-commerce, and shortage of inspectors and laboratories. The review recommends establishing a single food control/safety authority, developing more food safety laboratories, and adopting innovative technologies to detect and prevent food fraud. SA faces a serious food fraud crises unless decisive action is taken.

A decision analysis of cancer patients and the consumption of ready-to-eat salad.

Listeria monocytogenes is a foodborne pathogen of concern for cancer patients, who face higher morbidity and mortality rates than the general population. The neutropenic diet (ND), which excludes fresh produce, is often utilized to mitigate this risk; however, an analysis weighing the theoretical listeriosis risk reduction of produce exclusion aspects of the ND and possible negative tradeoffs has never been conducted. Consequently, this work constructed decision analytic models using disability-adjusted life years (DALYs) to compare the impacts of the ND, such as increased neutropenic enterocolitis (NEC) likelihood, with three alternative dietary practices (safe food handling [SFH], surface blanching, and refrigeration only) across five age groups, for cancer patients who consume ready-to-eat salad. Less disruptive diets had fewer negative health impacts in all scenarios, with median alternative diet DALYs per person per chemotherapy cycle having lower values in terms of negative health outcomes (0.088-0.443) than the ND (0.619-3.102). DALYs were dominated by outcomes associated with NEC, which is more common in patients following the ND than in other diets. Switchover point analysis confirmed that, because of this discrepancy, there were no feasible values of other parameters that could justify the ND. Correspondingly, the sensitivity analysis indicated that NEC mortality rate and remaining life expectancy strongly affected DALYs, further illustrating the model's strong dependence on NEC outcomes. Given these findings, and the SFH's ease of implementation and high compliance rates, the SFH diet is recommended in place of the ND.

Clinical Outcome and Factors with Dietary Behaviors in Pregnant Women with Listeria monocytogenes: A Hospital-Based Case-Control Study in Shanghai.

Listeriosis is a globally rare foodborne disease that causes fetal-placental infection, leading to adverse pregnancy outcome, while limited research among pregnant women is available in China. This study was therefore aimed at analyzing the incidence, clinical manifestations, and clinical outcome of listeriosis among pregnant women and its associated dietary behavior risk factors in prevention. A hospital-based case-control study had been conducted from January 2017 to December 2021. Clinical data, laboratory information, and questionnaires including dietary behaviors and personal hygiene were collected within 2 days after case diagnosis. There were 48 pregnant women, including 12 cases and 36 controls, with an average age of 31.19 ± 3.75 years. The incidence of admission-based listeriosis among pregnant women was 1.6058 per 10,000. The 12 strains were divided into 3 serotypes: 1/2a(83.33%), 1/2b(8.33%), and 4b(8.33%). Among the cases, 5 cases (41.67%) resulted in abortion, 3 cases (25%) induced preterm labor, and 4 cases (33.33%) had full-term deliveries after treatment. There were 7 live births in the case group, among which 6 were admitted to the neonatal intensive care unit (NICU), while 1 case had a healthy fetal outcome. All patients in the control group gave birth to live fetuses. Epidemiological investigation revealed that pregnant women dining at restaurants three or more times per week might increase the risk of having Listeria infection. There were no significant differences in dietary consumed behaviors, hand hygiene, and refrigerator usage behaviors between case and control groups. The study suggested that dining at restaurants might be associated with Listeria infection among pregnant women. Therefore, it is essential to enhance education on listeriosis serious consequences and promote healthy dietary and hygiene habits among pregnant women.

Listeriosis in Poland in 2012-2021. / Listerioza w Polsce w latach 2012-2021.

AIM: The aim of the study is to present and evaluate the epidemiological situation of listeriosis in Poland in the years 2012-2021. MATERIAL AND METHODS: The analysis material consisted of data from individual epidemiological case reports on listeriosis submitted to the Department of Epidemiology of Infectious Diseases and Surveillance of the NIPH NIH - NRI by state sanitaryepidemiological stations in the form of paper questionnaires (2012-2019) and in the electronic form through the EpiBaza system (2020 and 2021), as well as aggregated data from the bulletin "Infectious Diseases and Poisoning in Poland". RESULTS: Between 2012 and 2021, a total of 896 cases of listeriosis were registred in Poland. The median incidence was 0.23 per 100,000 population, which was an increase by 52.2% compared to the previous 5-year period (2007-2011). Every year, more than 90% of cases were hospitalized. The highest percentage of patients were in the age group >60 years old (65.5%). From 2012 to 2019 (in the years when information on cases was collected on a paper form), a total of 275 deaths of patients from listeriosis were recorded (38.4% of all reported cases). According to data from the EpiBaza system, in 2020 and 2021 there were 5 (8.33%) and 25 (20.83%) deaths due to listeriosis. A total of 92.1% of patients with listeriosis had significant predisposing factors for the occurrence of this disease, most of which were associated with neoplasia and heart disease and were present in half of all cases. As part of routine surveillance, no epidemic outbreak associated with Listeria monocytogenes infection was reported in Poland in the years 2012-2021.A total number of 49 pregnant women with listeriosis were reported during described period. Between 2012 and 2021, 37 cases of congenital listeriosis were reported. The median of incidence was 1.07/100 thousand live births, a decrease of 26% compared to the previous 5-year period (2007-2011). Of all congenital infections in newborns, 12 deaths (32.43%) were reported. CONCLUSIONS: The epidemiology of listeriosis is changing both in the EU/EEA countries and in Poland: the incidence is increasing and the distribution of cases in different age groups is changing, affecting primarily the elderly, especially those with predisposing diseases. Although 2020 tere was a decrease in the number of cases at EU level, possibly related to the COVID-19 pandemic, the overall trend of listeriosis cases isincreasing. The clinical condition has a significant impact on the course of L. monocytogenes infection: in healthy people, infection is usually asymptomatic. The disease primarily affects immunocompromised people. In contrast, infection of pregnant women can lead to premature birth, miscarriage, meningitis and neonatal sepsis with mortality rate of 20-30%. The growing trend in listeriosis is alarming and requires greater attention in terms of prevention and control of the disease.

Imaging modalities in neurolisteriosis: a literature review.

Listeriosis caused by Listeria monocytogenes due to its ability to withstand harsh conditions and form biofilms on post-processed food poses a serious public health issue. It typically manifests itself with fever and gastrointestinal symptoms, but it can escalate with life-threatening conditions, especially in immunocompromised patients, the elderly, oncologically sick, and even pregnant women. The diagnosis is based on blood and cerebrospinal fluid culture growth, but it presents significant challenges due to deceptive findings and low positivity rates, the golden standard includes molecular diagnostic tests such as real-time PCR and metagenomic next-generation sequencing, which offer higher sensitivity and rapid detection. Radiological imaging, particularly magnetic resonance imaging, can play a crucial role in diagnosis of central nervous system (CNS) invasion by L. monocytogenes, enabling the detection of characteristic CNS lesions. The aim of the paper was to sum up the imaging features of L. monocytogenes CNS invasions in conventional imaging techniques, which can potentially speed up the diagnostic workflow for patients presenting with neurological symptoms associated with L. monocytogenes infection, particularly when conventional tests yield inconclusive results.

Apolipoprotein E genotype affects innate susceptibility to Listeria monocytogenes infection in aged male mice.

Apolipoprotein E (ApoE) is a lipid transport protein that is hypothesized to suppress proinflammatory cytokine production, particularly after stimulation with Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). Studies using transgenic ApoE human replacement mice (APOE) expressing one of three different allelic variants suggest that there is a hierarchy in terms of responsiveness to proinflammatory stimuli such as APOE4/E4 > APOE3/E3 > APOE2/E2. In this study, we test the hypothesis that APOE genotype can also predict susceptibility to infection with the facultative intracellular gram-positive bacterium Listeria monocytogenes. We found that bone-marrow-derived macrophages isolated from aged APOE4/E4 mice expressed elevated levels of nitric oxide synthase 2 and were highly resistant to in vitro infection with L. monocytogenes compared to APOE3/E3 and APOE2/E2 mice. However, we did not find statistically significant differences in cytokine or chemokine output from either macrophages or whole splenocytes isolated from APOE2/E2, APOE3/E3, or APOE4/E4 mice following L. monocytogenes infection. In vivo, overall susceptibility to foodborne listeriosis also did not differ by APOE genotype in either young (2 mo old) or aged (15 mo old) C57BL/6 mice. However, we observed a sex-dependent susceptibility to infection in aged APOE2/E2 male mice and a sex-dependent resistance to infection in aged APOE4/E4 male mice that was not present in female mice. Thus, these results suggest that APOE genotype does not play an important role in innate resistance to infection with L. monocytogenes but may be linked to sex-dependent changes that occur during immune senescence.

Listeria monocytogenes Illness and Deaths Associated With Ongoing Contamination of a Multiregional Brand of Ice Cream Products, United States, 2010-2015.

BACKGROUND: Frozen foods have rarely been linked to Listeria monocytogenes illness. We describe an outbreak investigation prompted by both hospital clustering of illnesses and product testing. METHODS: We identified outbreak-associated listeriosis cases using whole-genome sequencing (WGS), product testing results, and epidemiologic linkage to cases in the same Kansas hospital. We reviewed hospital medical and dietary records, product invoices, and molecular subtyping results. Federal and state officials tested product and environmental samples for L. monocytogenes. RESULTS: Kansas officials were investigating 5 cases of listeriosis at a single hospital when, simultaneously, unrelated sampling for a study in South Carolina identified L. monocytogenes in Company A ice cream products made in Texas. Isolates from 4 patients and Company A products were closely related by WGS, and the 4 patients with known exposures had consumed milkshakes made with Company A ice cream while hospitalized. Further testing identified L. monocytogenes in ice cream produced in a second Company A production facility in Oklahoma; these isolates were closely related by WGS to those from 5 patients in 3 other states. These 10 illnesses, involving 3 deaths, occurred from 2010 through 2015. Company A ultimately recalled all products. CONCLUSIONS: In this US outbreak of listeriosis linked to a widely distributed brand of ice cream, WGS and product sampling helped link cases spanning 5 years to 2 production facilities, indicating longstanding contamination. Comprehensive sanitation controls and environmental and product testing for L. monocytogenes with regulatory oversight should be implemented for ice cream production.

Listeria monocytogenes Transmission from Donated Blood to Platelet Transfusion Recipient, Italy.

We report Listeria monocytogenes infection in a patient in Italy who was transfused with pooled platelet concentrate. Genomic analysis revealed that L. monocytogenes isolates from the donor blood unit, the transfused platelets, and the patient's blood culture were genetically closely related, confirming transfusion transmission. Additional surveillance and secondary bacterial screening could improve transfusion safety.

Listeria monocytogenes infection in pregnant macaques alters the maternal gut microbiome†.

OBJECTIVES: The bacterium Listeria monocytogenes (Lm) is associated with adverse pregnancy outcomes. Infection occurs through consumption of contaminated food that is disseminated to the maternal-fetal interface. The influence on the gastrointestinal microbiome during Lm infection remains unexplored in pregnancy. The objective of this study was to determine the impact of listeriosis on the gut microbiota of pregnant macaques. METHODS: A non-human primate model of listeriosis in pregnancy has been previously described. Both pregnant and non-pregnant cynomolgus macaques were inoculated with Lm and bacteremia and fecal shedding were monitored for 14 days. Non-pregnant animal tissues were collected at necropsy to determine bacterial burden, and fecal samples from both pregnant and non-pregnant animals were evaluated by 16S rRNA next-generation sequencing. RESULTS: Unlike pregnant macaques, non-pregnant macaques did not exhibit bacteremia, fecal shedding, or tissue colonization by Lm. Dispersion of Lm during pregnancy was associated with a significant decrease in alpha diversity of the host gut microbiome, compared to non-pregnant counterparts. The combined effects of pregnancy and listeriosis were associated with a significant loss in microbial richness, although there were increases in some genera and decreases in others. CONCLUSIONS: Although pregnancy alone is not associated with gut microbiome disruption, we observed dysbiosis with listeriosis during pregnancy. The macaque model may provide an understanding of the roles that pregnancy and the gut microbiota play in the ability of Lm to establish intestinal infection and disseminate throughout the host, thereby contributing to adverse pregnancy outcomes and risk to the developing fetus.

MicroRNAs induced by Listeria monocytogenes and their role in cells.

Listeria monocytogenes (Lm) causes abortions at high rates and threatens newborns' lives. Also, the elderly and immunocompromised individuals are particularly vulnerable neurologically. The bacterium exerts its pathogenesis intracellularly by manipulating cell organs. It manipulates nucleus elements, microRNAs (miRNAs), in order to increase survival and evade immunity. miRNAs are small non-coding RNAs that degrade gene expression post-transcriptionally. Any alteration to the expression of miRNAs affects various cascades in cells, especially immunity-related responses. Thus, utilizing miRNAs as a novel therapeutic agent not only restricts infection but enhances immunity reactions. This review provides an overview of miRNAs in listeriosis, their role in cells, and their prospects as therapy.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.