The study was carried out in Oda Bultum district of Western Harerghea Zone, Oromia Regional State; Eastern Ethiopia. The specific site was Charcher Oda Bultum Farmers Cooperative Union farm, which is found in Gode-Hora sub-district. Oda Bultum district is located at approximately 375 km far from Addis Ababa and 50 km from zonal capital, Chiro. Geographically this area has an altitude of 1400 - 3100 m.a.s.l and the specific location of the site is provided bellow (Figure 1). The area has a mean temperature ranging from 22˚C - 28˚C. It receives an average annual rainfall of 900 mm - 1200 mm with bimodal distribution of the seasonal pattern peaking in mid-April and mid-August of the year; however there is a variation from year to year [11]. Currently, the Farm is being operated with 200 local breed in fattening, 50 Cross breed and 140 Borena breed in dairy Farms. The Farm is equipped with production facilities like feed chopper, feed mixer, milking machine, basic veterinary equipment for clinical diagnosis and modern housing for both fattening and dairy which is suitable lay out for the purpose of the study.

In order to meet the specific objective, analogous to [12], the study was conducted in one commercial feedlot setting with two pens of 100 and 30 animals each. The Farm is found in the study area stated above. All the bulls of study subjects were zebu breed of those mainly produced by the local small holders. They were bought from the local markets Baddessa town surrounded by highlands whereas Boke, Gabiba and Milkae were from Wabi-Shebelle basin lowland areas of Harerghea where these three lowland areas are 30 km, 60 km and 120 km distance from the study Farm. The production system in low land is pastoral and in highland managed under zero grazing by the small holders [13]. The Chercher Oda-Bultum Farmers Union collect these animals for the purpose of finishing and supplying beef to abattoir or butchers of central Ethiopian markets like Addis Ababa, Mojo, Adama and large institutes including Haramaya and Oda-Bultum Universities.

For connivance of the following exposed and non-exposed animal at abattoir, all exposed animals and the randomized control groups, which were slaughtered at Haramaya University Abattoir, were examined for samples.

Body condition scoring [14], and age determination of the study animals were done according to the standards developed by Canadian Food Inspection Agency [15]. Both control and treatment cohorts of feedlot cattle were received a starter diet and a finishing diet during the feeding period. The treatment diets were differed from the control cattle diet by the addition of EM · 1^© inoculants (EM Research Organization Japan, Inc. #3600-01-007771) and the control group was used molasses as placebo as the color of two liquids are similar as well as used as owner blinding.

The product was supplied by EM-Woljejii Agricultural Industry PLC, which is accredited distributer in domestic market. They were supplied in the form of feed mixed with EM, molasses and warm water (chlorine free) in the ratio of 1:1:18 liter, according to manufacturer’s recommendation, with the target dose being 5 × 10¹⁰ cfu/day/head of Lactobacillus bacteria [16]. For 90,100 and 115 days based on batch of animals to be slaughtered. Treatment and control diets were administered for the duration of the feeding period and separate feeding trucks were used to administer the two different diets. Other than the treatment feed for treatment groups, the rest were the same in terms of natural challenge and local feed including hay, teff straw and “frushka”, coffee husk. Close supervision and monitoring were in place by using tools like checklists.

Double blinded parallel-group-designed and randomized controlled field trial (RCFT) were conducted in which the treatment EM were supplemented to treatment group (n = 100) and control (n = 30) animals in each pen. Within pens the animals were clustered based on their biological differences considering animal age determination [15], body condition [14], body weight, sources and exposure time. Hence, all animals were tagged and registered for onset of the study. For these purpose, animals’ attendants and employed workers were trained on how to prepare and feed EM-microbial inoculants.

Following the study animals at study abattoir in both cohorts of the study, one sample of SLN and one sample MLN per carcass were collected from both treatment and control groups of animals at study abattoir immediately after slaughter. Thus, 130 samples of LNs each from both groups of animal (N = 260) were collected for laboratory examination. The cattle were slaughtered in three groups and therefore housed at the feedlot for 90, 100, and 115 days of exposure, respectively based on national fattening package [17]. Thus, pair of samples (SLN and MLN) from each the sampled animal were collected aseptically and separately.

The Farm was sampled by convenience sampling as it was accessible, manageable and convenient for group harvesting and sample collection within the project time frame.

The prospective randomized control field trial in parallel-groups-designed study based on feed supplemented by EM and non-EM supplemented group. Sample size was calculated by using the formula given by [18], which is appropriate in comparison of effects.

N = 0.25/SE²,

where: N = sample size, SE (standard error) = 5%; Hence, the required sample sizes were (n = 100) for treatment and (n = 30) animals for control cohort. Assuming that, confidence interval (CI) = 95%; desired absolute precision (α) = 0.05; Power (P) = 96%.

Following specific identification given during the feeding, the samples of SLN and MLN were aseptically collected and registered with same identification code used while animals were alive at the farm. On 90^th day (n = 42), 100^th day (n = 40) and 115^th day (n = 48) animals were slaughtered from which (n = 84), (n = 80) and (n = 96) samples were collected separately on 90^th, 100^th and 115^th days respectively. The samples were transported to Veterinary Microbiology Laboratory, College of Veterinary Medicine of Haramaya University for immediate process on the date of sampling. Sample collection and processing were done aseptically (flaming the sampled LNs before processing) but blinded using the coding system that has been given at the beginnings of study. Thus, codes were lifted in to Excel sheet after data collection in order to conduct statistical analysis.

Sampled LNs were processed as previously described [12] [19]. Surrounding fat and fascia were trimmed from LN samples, which were weighed, surface sterilized by surface flaming, placed into individual filtered sample bags, and pulverized using a stomacher (model: 400 stomacher, Seward, Worthington, UK) at 230 rpm for 2 minutes. The isolation and identification of Salmonella were undertaken following conventional cultural methods. Briefly, each processed sample was pre-enriched in BPW (BM020, Sisco Research Laboratories; India), (1: 9) and incubated for 16 - 20 h at 37˚C. From the pre-enrichment broth, 100 micro liters were transferred into 9.9 ml of Rappaport Vassilliadis (RV) (Oxoid) broth and incubated at 42˚C for 24 h. A loop full of the inoculums from RV was streaked side by side onto Xylose Lysine Deoxycholate agar (XLD) (M031-500G, HiMedia Laboratories Pvt. Ltd.), and brilliant green phenol red lactose sucrose (BPLS) (Merck) agar plates and incubated at 37˚C for 24 h. The Presumptive Salmonella colonies were purified on fresh nutrient agar (HiMedia, India) and further characterized using conventional biochemical tests. Isolated Salmonella colonies were inoculated onto triple sugar iron agar (TSI) (M021-500G, HiMedia Lab. Pvt. Ltd., India) lysine iron agar (LIA) (CM081, Oxoid Ltd., England) Simmon’s citrate (M099, HiMedia Lab. Pvt. Ltd., India) and Moreover, two or more colonies from pure isolates were inoculated on urea broth (SRL, India) and incubated at 37˚C for 24 h for confirmation according to [20].

Raw data were interred to Microsoft Excel 2007^© and analyzed using STATA 12.1. For qualitative data, r/n (events/trials) binomial response variables was created for each control and treatment groups, where r is the number of positives and n is the number of lymph nodes to be assayed. Cohort study risk ratio was used grossly at pen level relative risk estimation. Multi-level-mixed effects Model, extension to Mixed-effects linear regression was constructed in which risk factors were considered a random variable and used to screen the potential confounding factors. Model estimation was achieved using maximum likelihood method and Wald Chi². To account for potential within and among pen dependency residuals (i.e., clustered outcomes) a random effect regression model was used. The mean prevalence for the treatment by time interaction was analyzed and data were used for estimation of percent efficacy. Relative risks (RR), relative risk reductions (RRR) and absolute risk (AR) at 95% confidence intervals (C.I. 95%) were calculated where (p < 0.05) was considered as significant Salmonella prevalence.

The total number of lymph nodes (N = 260), where, n = 130 and 130 of them were MLN and SLN respectively; 200 were collected from 100 cattle administered EM2 (Treatment) and 60 were collected from 30 cattle in the control group. A total of 33/100 (33%) and 21/30 (70%) LNs collected from cattle in the treatment and control groups were foundSalmonella positive respectively. A greater percent of positives (70%) were observed LNs collected from cattle in the control group (Table 1).

A significant reduction in Salmonella prevalence in LNs (MLN and SLN) was observed from cattle administered EM (p = 0.000). The risk of Salmonella harborage in LNs of EM supplemented group was 53% less than the counterpart and 37% of the risk could be reduced by EM supplementation (Table 1). On a percentage basis the amount of positive animals in the treatment group was 33%. A higher percentage of positives 70% were observed in cattle from control group. These results agree with the slight difference in treatment group from similar study by [13], which reported prevalence of 57% and 76.3% in treatment and control respectively.

In this study relative reduction in the prevalence of Salmonella within MLN and SLNs was observed in the study cattle presented for harvest after treated by EM. The data reported herein indicate that administering EM to cattle during the feeding period has an effect in reduction of Salmonella detected in MLN and SLNs of beef cattle. These preliminary data are impactful for the beef industry as well as public health, since lymph nodes, including SLNs within beef are commonly incorporated into beef trim destined for ground beef production.

As shown in (Table 2), variable proportions of Salmonella were observed in MLN and SLN of both control and EM-feed animals except for those EM-feed for 115 days those with good body condition. As age of animals increases the prevalence of Salmonella become increasing in both MLN and SLN of control groups but reduction in SLN of EM-feed animals were observed. Significant reductions in Salmonella prevalence with increase in treatment time for EM-feed group were observed.