Detection of Listeria species, factors associated, and antibiogram of Listeria monocytogenes in beef at abattoirs, butchers, and restaurants of Ambo and Holeta Towns, Ethiopia

Listeriosis is one of the globally distributed foodborne diseases with the highest fatality rate. Few studies were done on the occurrence of Listeria species from meat at abattoirs, butchers, and restaurants in Ethiopia, and there has been no study conducted at Ambo and Holeta town. The objectives of this study were to isolate and identify Listeria species, assess factors for contamination of meat, and antibiogram of Listeria monocytogenes along the meat chain in Ambo and Holeta towns, Central Ethiopia. AMK-Amikacin, AMP-Ampicillin, AMX-Amoxycillin, CIP-Ciprooxacin, CHL-Chloramphenicol, CLI-Clindamycin, CTX- Cefotaxime, GEN- Gentamicin, ERY- Erythromycin, NAL-Nalidixic Acid, NIT-Nitrofurantoin OXA- Oxacillin, PEN- Penicillin, TET-Tetracycline, VAN- Vancomycin, XST- Trimethoprim sulfamethoxazole, molecular on


Introduction
Foodborne bacterial infections describes the adverse health effect associated with eating of contaminated foods with pathogenic bacteria, including those originated from meat and that result either in morbidity or mortality worldwide in general and in developing countries in particular [1]. Meat is the most valuable food of animal origin and its chemical composition makes it one of the most vulnerable vehicles of infections agents [2]. Even though the association of the raw meat consumption habits with health impact over several years has little attention, consumption of raw meat in Ethiopia is deep-rooted tradition [3][4][5]. Consumption of raw meat contaminated with pathogenic Listeria species causes foodborne listeriosis [3,4,6]. Foodborne listeriosis is one of the important diseases affecting human health globally related to the increasing global trade and travel [4,7,8]. Thus, even though foodborne listeriosis may be comparatively rare, it causes severe and life-threatening infection in immunocompromised groups such as HIV patients, pregnant women, neonates and elderly [9].
Based on the phenotypic and genotypic characteristic similarities and differences, Listeria species are grouped in to 'Listeria sensu lato' and 'Listeria sensu strictu' [10,11]. Listeria sensu strictu is composed of L. monocytogenes, L.inoccua, L. seelgerii, L.welshimeri and L. marthi. All Listeria sensu strictu are catalase-positive, motile at least at 30 o C and grow below at 4 o C and gram-positive. Listeria sensu lato is composed of 11 species comprising L. grayi as well as L. eischmannii, L. oridensis, L. aquatica, L. newyorkensis, L. cornellensis, L. rocourtiae, L. weihenstephanensis, L. grandensis, L. riparia and L. booriae [12]. Listeria monocytogenes causes foodborne disease and consequently, a serious health problem, because of severe symptoms and high mortality. Nowadays, other species of Listeria are also known to produce a disease in humans and animals [13].
Risk factors of food contamination are de ned to be all the factors necessary for food contamination / foodborne outbreaks, infections [14]. Contamination of meat by microorganisms occurs in abattoir during slaughtering and spread from the intestinal tract and the exterior part of animals. Moreover, it can be contaminated in retailer shop and kitchen from air, workers, knives, cloths, carts, and refrigerators [15]. Poor food handling and sanitation practices, inadequate food safety laws, weak regulatory systems, lack of nancial resources, improper storage, poor personal hygiene during preparation, extended shelf-lives refrigeration, inadequate cooling and reheating create a favorable condition for the spread of foodborne etiologic agents [7,16,17]. Sociodemographic factors, worker's food safety and hygiene information, knowledge on food safety and hygiene, food safety principles and practices, food source and others are few of the food contamination predictors [14]. The increased use of antimicrobial agents in food animal production and human is a signi cant factor in the emergence of antimicrobial resistant bacteria The repetitive use of antimicrobials in food animal production for treatment and as growth promotion is signi cant factor for the emergence of multi drugs resistant strains [4,8]. Meat is a major source of transmission of antimicrobial resistant microorganisms to humans [18]. As a result, control and treatment of listeriosis is di cult and very hazardous without antimicrobial resistance interventions [19] There have been few studies conducted on the occurrence of Listeria species in food samples including meat sources like poultry, mutton, pork, seafood and other foods of animal origins in Ethiopia [3,4,7,20,21]. However, the occurrence, risk factors and antibiogram of Listeria species along the consecutive meat chain from abattoirs, butchers and restaurants have not been studied so far. Careful investigation of raw meat samples collected from the three spots of the beef chain (abattoir, butcher, and restaurant) will help to identify the weak links contributing to the contamination. This helps considerably for subsequent interventions in an attempts to protect consumers from foodborne illnesses and to reduce economic losses due to food spoilage. Therefore, the objectives of the present study were to isolate and identify Listeria specie, assess factors contributing for contamination of beef and determine the antibiogram of Listeria monocytogenes isolated from abattoir, butchers and restaurant in Ambo and Holeta towns, central Ethiopia.

Study area
The study was conducted in Ambo and Holeta towns found in Oromia Region, central Ethiopia. Ambo town is the administrative center of West Shoa Zone located 114 km West of Addis Ababa at the latitude of 8°59′N 37°51′E and longitude of 8.983°N 37.85°E. The elevation of Ambo town ranges from 1900 to 2275 meters above sea level (masl). Its temperature ranges from 19 o C to 29 o C with an average annual temperature of 22 o C and an average annual rainfall of about 900 mm. The town has a total human population of 74, 843 out of which 39,192 are males and 35,651 are females [22]. There are 46 legal butchers with their annexed restaurants and one municipality abattoir in Ambo town.
Holeta is located in Fin ne special zone, 44 km West of Addis Ababa with latitude and longitude of 9 o 3'N38 o 30'E/9.050'N38.500 o E. Its elevation is 2400 masl. It receives 1144 mm annual average precipitation, with an average minimum and maximum temperature of 6ºC and 22ºC, respectively. The total human population of Holeta town is 25,593, of whom 12,605 are men and 12,988 women [22]. There are 20 legal butchers with annexed restaurants and one municipality abattoir in Holeta town.

Study Population
All abattoirs, butchers, restaurants in Ambo and Holeta towns constituted the targets of this study. Cattle slaughtered in Ambo and Holeta abattoirs and beef sold in butcher shops and restaurants of Ambo and Holeta towns are the study population.

Study Design
The study was conducted between October 2017 to April 2018 in abattoirs, butchers and restaurants of Ambo and Holeta towns.
First, the list of all the current legal butchers, restaurants and abattoirs registered in Ambo and Holeta towns was collected from the towns' municipality (sampling frame) and then the two abattoirs (one from each town) and those butchers with annexed restaurant for handling meat were purposively identi ed.

Sample Size Determination
The required sample size was determined using a 95% con dence limit and a 7% sampling error and an expected prevalence of 25% [4] by the following formula [23].

Sample collection
Systematic random sampling method was used to select the animals from study population. Following slaughtering, meat samples were collected from three spots of the beef chain (abattoir, butcher, and restaurant) from the same animal. First composite meat samples of about 250 gm were collected from slaughtered cattle from four sites (ramp, ank, brisket, and neck) just after the stage of evisceration. The same procedure was followed on the second sampling spot, i.e. butcher shops. On the third sampling spot, 250 g raw meat that is prepared to be served for consumption (Kitfo-a traditional Ethiopian dish made from minced raw beef, chili, spice and butter blend or Tire segaspecial meat cut served raw) was purchased. All samples were collected aseptically, and the samples were labeled with necessary information including the sample code, date of sampling and sampling place (spot). Samples were kept in separate sterile plastic bags (Seward, England), to prevent spilling and cross-contamination and immediately transported to the Ambo University Veterinary Microbiology Laboratory in a cooler icebox with ice packs and processed within 4 hrs.

Isolation and identi cation of Listeria species
As per the recommendation of ISO [24], 25 g of sample was homogenized in 225 ml of Listeria enrichment broth (LEB) containing supplements (HiMedia, India) using a laboratory blender and incubated at 30 o C for 24 hrs. Then after 0.1 ml of mixed inoculum was inoculated into a tube containing 10 ml Modi ed Fraser broth (MFB) and incubated for 24 hrs at 37 °C. Following enrichment procedure, the inoculum was streaked on Oxford agar (OXA) containing manufacturer's supplements and the plates were incubated at 37 o C for 24-48 hrs. After 24 hrs of incubation, the growth of Listeria species on the Oxford agar plate was examined for black halo colonies of Listeria species, which remain the same after 48 hrs of incubation but with a sunken center.
The presumptive colonies were picked up and further puri ed on Tryptone Soya Yeast Extract agar (TSYEA). Subsequently, pinpoint colonies on TSYEA were subjected to identi cation procedures, which included Gram's staining followed by a microscopic examination, catalase test, and oxidase test. The characteristic Gram-positive, coccobacillus or short rod-shaped organisms, which were catalase-positive and oxidase negative, were sub-cultured in Brain heart infusion (BHI) broth at 25 °C for 12-18 hrs. Subsequently, the cultures showing typical tumbling motility were considered as "presumptive" Listeria isolates, which were in turn subjected to detailed biochemical tests viz.; CAMP test, methyl red, Voges-Proskauer, and sugar fermentation tests with xylose, rhamnose, and mannitol for identi cation of Listeria to species level.

Questionnaire Survey
A semi-structured pretested questionnaire survey and observational checklist were used on abattoir personnel, butcher shop workers and restaurant chefs and waiters to assess their hygienic practice in processing and handling beef and beef products along the chain. The independent factors investigated along with their categories include: sex (male vs. female), marital status (single vs. married), the residence of origin (rural vs. urban), educational status (uneducated, primary school, secondary school, and tertiary), time on work per day (≤ 8 hours vs. ≥ 9 hours), information on food hygiene, training on food safety, use of refrigeration, presence of insects and presence of rodents (all yes or. no), sanitation of butchers and restaurants, hygiene of the slicing materials, hygiene of the cutting boards, hygiene of the weighing balance and food handling surfaces (all categorized as poor, fair, good, and very good).
Then, a sterile cotton swab was used to spread the bacterial suspension on the Muller Hinton agar (HiMedia, India). According to the standard procedure CLSI [25], the disks were rmly placed in the interval of 3 cm spacing from each other onto the medium with sterile forceps and then incubated at 37 o C for 24 hrs. Then, the diameter of clear zones around the disks was measured with a ruler against black background and compared with standards given by CLSI [25]. L. monocytogenes ATCC7644, E. coli ATCC25922, and S. aureus ATCC6538 reference strains were used as quality control.

Data Management And Analysis
The collected data were entered into Microsoft Excel spreadsheets (Microsoft Corporation) and analyzed using STATA version 14.2 software (Stata Crop. College Station, USA). Descriptive statistics was utilized to summarize the occurrence, socio demographic characteristics of the respondents and antimicrobial susceptibility data using percentages. Univariable and multivariable logistic regression analyses with their Odd Ratio (OR) and 95% con dence Intervals (95% CI) were used to assess the factors associated with contamination of beef by Listeria species. During analysis, for all of the risk factors, the rst category of the independent variables (with the lowest percentage) was considered as a reference category. Non-collinear variables were selected with the help of a multicollinearity matrix. Elimination of collinear variable with the poor biological background to explain possible contamination of raw beef by Listeria species was used to handle collinear variables. Non-collinear variables that possessed a p-value of < 0.25 in univariable analysis were entered into the multivariable analysis. The model was constructed by a backward exclusion method. Hosmer-Lemeshow goodness of-t-test was used to assess the model tness. The reliability of the tted model was further evaluated using the receiver operating characteristics curve (ROC). The 95% con dence interval (CI) was used in all cases and the results were considered signi cant at p < 0.05.

Isolation and identi cation of Listeria species
Out of the total 450 meat samples examined, 128 (28.44%; 95%: CI: 24.32-32.86%) were positive for Listeria species. The occurrence of Listeria species in Ambo town (20.66%) was much lower when compared to Holeta town (60.92%).The highest rate of occurrence of Listeria species was recorded in restaurants (30.00%), followed by butchers (29.33%) and then abattoir (26.00%). The occurrence of Listeria species in abattoir and restaurant samples of Holeta town was relatively higher than abattoir and restaurant samples of Ambo town (Table 1). Out of the total 128 Listeria species isolated, a high contamination rate of Listeria grayi and low contamination rate of Listeria seeligeri were recorded. The overall occurrence of L. monocytogenes was 4.44% ( Table 2). The occurrence of L. monocytogenes in abattoirs, butchers, and restaurants raw meat samples of cattle were 33%, 1.56%, and 1.56%, respectively.

Risk Factors
Socio-demographic characteristics, knowledge, and practice on food safety and hygiene All respondents from abattoirs and butchers were male while all respondents from restaurants were females. The majority of the respondents from abattoirs (57.58%) were within the age group of 18-24 years while that of butchers (58.62%) and restaurants (52.41%) were ≥ 35 years and 25-34 years, respectively. The majority of the respondents attended primary education and had less than one year of work experience. Most of the workers in butchers and restaurants work ≥ 9 hours per day (Table 3). FrEq. = Frequency.
The vast majority of respondents had information/knowledge on food hygiene and safety, often without practicing it but had no training on food safety (Table 4). Univariable logistic regression analysis also showed that the occurrence of Listeria species in meat was signi cantly associated with the hygiene of cutting boards (p < 0.05). All the other factors investigated did not show signi cant association with the risk of cattle raw meat contamination (p > 0.05) ( Table 5).  not entered into the multivariable model, although hygiene of the slicing material vs. sanitation of butchers and restaurants has more biological ground to explain raw meat contamination by the Listeria species. Multivariable logistic regression analysis revealed that study town and season are independent predictors of contamination of meat by Listeria species (p < 0.05). Information on food hygiene and safety (p = 0.054), and the number of hours worked per day ((p = 0.079), though not signi cantly associated with the prevalence of Listeria species, they are close for association (Table 6).

Antimicrobial Susceptibility
Out of the 20 L. monocytogenes isolates subjected against 16 commercial antimicrobial discs. 16 isolates (80%) were resistant to oxacillin; 14 (70%) were resistant to amikacin and nalidixic acid; 12 (60%) were resistant to chloramphenicol, 11 (55%) were resistant to tetracycline. The L. monocytogenes isolates were 95%, 90% and 85% susceptible to amoxicillin, vancomycin, and clindamycin, respectively (Table 8). All identi ed Listeria monocytogenes had developed resistance to two or more classes of antimicrobial drugs. Nineteen (95%) L. monocytogenes isolates were resistant to three or more classes of antimicrobial (multidrug resistance-MDR). One isolate (5%) had developed resistance to ten classes of antimicrobials. The most common MDR pattern was observed against amikacin, ceftoxamine, nalidixic acid and Oxacillin. Multidrug resistance of L. monocytogenes isolates, number of isolates and percentages resistance as shown below (Table 9).

Discussion
Occurrence of Listeria species in raw beef from abattoir, butcher and restaurants In the present study, Listeria species was isolated from 28.44% of raw beef samples from abattoir, butcher, and restaurants which agrees with the reports from Addis Ababa (27.5%) [3]. Other studies have reported signi cantly higher isolation rates of Listeria species in raw meat such as 95% in Brazil [26], 81.5% in Turkey [27], 58% in Nigeria [28], 54.1% in Turkey [29], 51.3% in Ethiopia [21], and 50% in Jordan [30].
The 4.44% isolation rate of L. monocytogenes in this study was comparable with the 4.1% from goat meat in Ethiopia [7] and 4.1% in raw beef in Ethiopia [3]. However, the current nding was higher when compared to the 1.29% isolation rate from raw cow and goat meats [31], 1.6% from minced beef [20] and 2.5% from bovine carcasses in Poland [32]. The present nding of L. monocytogenes (4.4%) was lower when compared to the 15.6% [33] in Nordic countries,15.4% in Bangkok [34], 25.5% in Turkey [27]. The relatively low prevalence of L. monocytogenes in present study might be attributed to the difference in the study season, geographic conditions, and sample size [35,36].
Meat can be contaminated by L. monocytogenes at abattoirs by cross-contamination during slaughtering, evisceration, and other processing steps. Additionally contamination and growth of Listeria at the next chain (butchers and restaurants) could occur due to poor hygienic handling and processing refrigeration; and the suitability of the meat pH, water activity, and nutrient content [37]. The isolation rate of other Listeria species in the present study ranged from 1.78% − 10.22%. The 2.22% isolation rate of L. ivanovii in the present study is by far lower than the 21.9% reported by Al-Nabulsi et al [16] and 19.8% by Alsheikh, Mohammed and Abdalla [38] reported from various types of meat and meat products. However, the isolation rate of L. ivanovii in the present study is comparable to 2% reported from raw meat in Ethiopia [4]. The 3.77% isolation rate of the L. welshimeri in this study is in agreement with the 2.75% [16], and 4% [4] reported previously. On the other hand, the 1.78% isolation rate of L. seeligeri in the present study disagrees with the 27% isolation rate of L. seeligeri by Al-Nabulsi et al [16] but it is in line with the 1% and 2% isolation rate of L. seeligeri previously reported by [38] and [4], respectively. The lower occurrence of L .ivanovii, L. welshimeri and L. seeligeri in the present study might be linked to the hygienic status of food processing environments and the differences in the bacteriological detection methods [4,38,39].
Most research reports indicate that L. inoccua is the most common species isolated from different meat samples. For example, in Ethiopia, 83% [3] and 19% of L. innocua [4] have been reported. But, in this study, L. grayi (10.22%) and L. inoccua (6.22%) were more common than the other Listeria species. Few reports from meat samples showed that L. grayi was the second most abundant Listeria species next to L. ivanovii [16]. The relatively high rate of L. grayi (10.22%) and L. inoccua (6.22%) isolation in the current study is in line with the results of Fissiha [40] in Ethiopia. The abundance of L. grayi and L. inoccua in the present study might be related to the species entrance to the processing environments via as a part of intestinal microbial ora, improper hygienic practices during processing and food handling [41,42].

Risk Factors Assessment
The occurrence of Listeria species in cattle raw meat was nearly 9 times higher in the dry season when compared to the wet season. The signi cantly high rate isolation of Listeria species in cattle raw meat during dry season might be due to the large sample size (more than 75% of the meat samples) in the dry season and the environmental stress in cattle during the dry season that led to the shedding of the organisms in feces. It is well known that a dry climate harms the persistence of Listeria species [43]. Thus, drying is linked to water activity lowering of meat and meat products. As a consequence, the lowered moisture content of the meat enables the organism to persist in (resist) high temperature [44]. In addition, the shortage of water during dry season might impair hygienic activities of personnel and equipment in the establishments thereby increasing the chance of contamination. The isolation of Listeria species from meat in both dry and wet seasons suggests the existence of natural reservoirs of the bacteria in cattle or the environment. On the other hand, the signi cantly high occurrence of Listeria species in butchers and restaurants where the employees work ≥ 9 hrs as compared to those working ≤ 8 hrs might be related to the change in the hygienic behavior of works as they get exhausted/tired. This is the rst report indicating that employees working ≥ 9 hrs per day in handling beef are important source of beef contamination in the study areas.
In the present study, the place where the study was carried out was the predictor of Listeria species isolation (p ≤ 0.001). The signi cantly high recovery rate of Listeria species from Holeta (57.14%) as compared to Ambo (21.37%) town might be related to the cool temperate climate of Holeta town favorable for the survival and multiplication of the organisms.
This study showed signi cant association between the risk of cattle raw meat contamination by Listeria species and information on food hygiene and safety. This nding is inconsistent with the reports of E De Booek et al [45] who suggested a signi cant contribution of the lack of knowledge on food hygiene and practice for high risk of contamination. In contrary to this, the results of the present study showed that less adoption of information on food hygiene and practice as a habit or culture every time during working hours by employees or workers. Thus, it may contribute to the high risk of cattle raw meat contamination by Listeria species. Moreover, the absence of law about HACCP (Hazard Analysis Critical Control Point) programs, its implementation, and the widespread inadequate hygienic practices in Ethiopia coupled with only having knowledge on food hygiene and safety without its practice or implementation might be regarded as a contributing factor for meat contamination.
Univariable logistic regression analysis also identi ed the hygiene of cutting boards as a risk of cattle raw meat contamination by Listeria species. This is in line with the ndings of previous workers [46][47][48]. The types or quality, poor hygiene and absence of frequent sanitation of the cutting boards and use of unclean water for washing cutting boards might be linked to a higher chance of cutting boards being contaminated with Listeria species [48].
Antimicrobial susceptibility pro le of the L. monocytogenes isolates The highest percentage of resistance was noted for oxacillin (75%) followed by 55-70% resistance to amikacin, nalidixic acid, tetracycline, and chloramphenicol. The 75% resistance of oxacillin in the present study is comparable with the72.2% resistance reported by Wieczorek [32] and [49]. While, the 70% resistance to amikacin is in contrary to the 100% susceptibility reported by Indrawattana [34]. The resistance against nalidixic acid and tetracycline in this study are in accord with the reports of Maktabi et al [35].
The 30% resistance to cipro oxacin of this study is lower compared to the 44.4% [50] and 56% resistance [51]. The 30% resistance to erythromycin and trimethoprim-sulfamethoxazole disagrees with 87.5% [51] and 69.4% [50] resistance. Although 35% resistance to trimethoprim-sulfamethoxazole and gentamicin in the present study agrees with the 24% [35] and 36.1% [50] resistance, it contradicts with the 87.5% trimethoprim-sulfamethoxazole and 72.21% gentamicin resistance [51]. The 15% resistance to clindamycin is in line with the 12% resistance reported by Maktabi et al [35]. However, the 15% of cefotaxime and 25% penicillin resistance in this study is highly divergent from the 77.5% cefotaxime and 66.7% penicillin resistance reported from Ethiopia [4]. The low level of resistance (5-15%) to amoxicillin, ceftoxamine, vancomycin and clindamycin in this study might be due to absence of usage of these drugs in veterinary medicine in Ethiopia which plausibly suggests that these antimicrobials remain an alternative regimen against the organisms [4,52]. While the relatively high resistance, (≥ 30%) observed in erythromycin, chloramphenicol, trimethoprim-sulfamethoxazole, oxacillin, tetracycline, gentamycin, and trimethoprim-sulfamethoxazole might be related to the more frequent or improper usage, particularly in the public health sectors. Nevertheless, the study warrants frequent surveillance on the change in the pattern of antibiogram for this organism.
The present rate of MDR L. monocytogenes (95%) is higher when compared to the 72.3% MDR identi ed from raw foods [53]. In agreement with the present study Odu and Okonko [8] also reported L. monocytogenes isolates that are 100% MDR. The MDR patterns of L. monocytogenes in the study towns might be due to the non-prescribed frequent and non-judicious usage of antimicrobials in livestock and public health sectors in the study towns [4]. The MDR to L. monocytogenes may occur due to plasmid or chromosomal genes transfer and mutation events in chromosomal genes from other Listeria species and Gram-positive bacteria, which may be found in foods [53,54].
The high percentage of L. monocytogenes isolates resistant to the relatively cheaper and commonly available antimicrobials is worrisome as it might lead to the use of mandatory and more expensive drugs [4]. This is a problem for listeriosis high-risk groups in developing countries like Ethiopia because of the high cost of hospitalization and recent drug treatment leads to an economic burden on families and societies.
The isolation of L. monocytogenes from raw meat should be considered as a microbiological hazard for people since the consumption of raw or undercooked meat in the study area is a widespread food habit.
The present ndings have also great implications for the public health in Ethiopia because of the high fatality of listeriosis, the abundance of immunocompromised people and inadequate hygiene and awareness of the community.
The limitations of this study include the following: Firstly, due to the lack of resources and facilities, serotyping and molecular works on Listeria species have not been done. Secondly, antimicrobial susceptibility testing was performed only for L. monocytogenes. Thirdly, samples from meat handlers, equipment, and the environment were not taken to see their association. Fourthly, the questionnaire data collected from abattoir workers was not included in the logistic regression analysis due to the small number of workers (n = 33) who responded to our questions and the di culty of linking responses of workers to a particular sample result as all worker handle the carcasses. This makes comparison of the results form abattoirs with that of butchers and restaurants hard.

Conclusions
The study con rmed that contamination of raw meat sold for human consumption by Listeria species is widespread in the area. Six Listeria species (Listeria monocytogenes, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, Listeria innocua and Listeria grayi) were identi ed. There overall occurrence of Listeria species was medium while the isolation rate of L. monocytogenes was relatively low. The study towns, season and working hours per day are independent predictors of Listeria species isolation. The high antimicrobials resistance and multidrug resistance among L. monocytogenes isolates are of great public health importance. Amoxicillin, clindamycin, nitrofurantoin, and vancomycin might be good drugs for the treatment of L. monocytogenes infections in the study areas. Therefore, regular training should be given for worker in the establishments and follow up on the prudent use of antimicrobial drugs in veterinary and public health sectors are important; and subsequently, further serological and molecular studies on Listeria species are proposed.

Declarations
Availability of data and materials The raw datasets used during the current study can be obtained upon the reasonable request of the corresponding author. The questionnaire used during the current study is available as Additional le 1.

Ethics approval and consent to participate
All study subjects were informed about the study and written informed consents were obtained from all the owners and workers of the Ambo and Holeta town butchers, restaurants and abattoirs. Con dentiality was assured by using codes. Ethical clearance was obtained from the Ambo University research and ethical review committee.

Consent for publication
Not applicable Funding This work was supported by Ambo University. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Authors' contributions EZG conceived the idea, designed the project, supervised the work, analyzed and interpreted the data and drafted and approved the nal manuscript for publication. GA performed the laboratory tests, participated in drafting the article and in approval of the nal version for publication. BMB, EJS and HAA designed the project, supervised the work, revised the manuscript and approved the nal article for publication. LMM and NDT participated in data collection, eld and laboratory supervision, gave comments on the manuscript and approved the nal version for publication.