Food-borne diseases are a public health problem in developed and developing countries. More than 250 different food-borne diseases have been described. Most of these diseases are infections caused by a variety of bacteria, viruses and parasites. Salmonella has been one of the most commonly reported causes of food-borne pathogens from distant and recent times (Pui et al., 2011; Hoffmann et al., 2012). According to a recent study (Kirk et al., 2015) commissioned by the WHO on the global disease burden of food-borne diseases in humans, food-borne illnesses from diarrheal and invasive non-typhoidal Salmonella enterica, resulted in the largest disease burden, highlighting the significant public health importance of Salmonella infections and the urgency for control, particularly in low-and middle-income countries where most burden of diseases and occurrence of mortality cases are reported. Salmonella species belong to Gram-negative, rod shaped, facultative intracellular bacteria that successfully infects a wide variety of hosts. Salmonella is comprised of two species, Salmonella bongori and S. enterica (Guibourdenche et al., 2010). Out of these 2,700 serovars, nearly 1500 belong to the S. enterica subsp. enterica. Serovars of the enterica sub species can be divided into three groups depending upon their ability to infect a wide variety of hosts: Serovars which have a broad host range also called as unrestricted serovars as these infect nearly all animals and pose a greater zoonotic potential than their other counterparts, Serovars which accidentally infect hosts other than their most adapted or preferred (McCuddin et al., 2006; Shahen et al., 2019), and serovars which are restricted to one specific host only. Salmonella transmits to humans can occur through several routes. these are  consumption  of  contaminated  food  products  (milk,  eggs, and meats), direct contact with animals and their environment, cross-contamination  through  direct  contact  of  foods  to  contaminated surfaces  such  as  stainless  steel,  hanging  material,  knife,  bucket  where milk are collected are a key mechanism for pathogens to contaminate food  products (Kusumaningrum et al., 2003; Uddin et al., 2017). 

Excretion of Salmonella with faces can contaminate water, soil, other animals and feed. Although Salmonella primarily  intestinal  bacteria,  due  to  its  ubiquitous  nature common in the environment and commonly found in farm  effluents human sewage and in any materials subject to faecal contamination as a  result  it  leads  the  contamination  of  milk  and  meat  products  to originate  either  from  infected  live  animals  or  from  cross-contamination while during processing. Despite the controls that have already been put into place, Salmonella infection arising from contaminated food continues to be an immense problem with millions of cases occurring annually throughout the world. In addition to the misery caused, financial loss is enormous (Hendriksen, 2003). An increasing proportion of Salmonella isolates is resistant to commonly used antibiotics in both developing and developed countries (Threlfall, 2002), and this increase is seen in both veterinary and public health sectors (Kemal, 2014; Van Boeckel et al., 2015).

Using antimicrobial agents for cattle have been implicated as a source of human infection with antimicrobial resistant (AMR) Salmonella through direct contact with livestock and consumption of raw milk, meat and contaminated materials (Alexander et al., 2009; Sharif et al., 2019). Antimicrobial resistant Salmonella are increasing due to the use of antimicrobial agents in food animals at sub-therapeutic level or prophylactic doses for growth promotion and markedly increase the human health risks associated with consumption of contaminated milk and meat products (Zewdu and Cornelius, 2009; Md et al., 2006), through mutation, acquisition of resistance encoding genes (Fluit, 2005) and irrational use of antimicrobials in food animals (Fluit, 2005; Beyene and Tesega, 2014).

In Ethiopia, as in other developing countries, it is difficult to evaluate the burden of food-borne diseases, because of the limited scope of studies and lack of coordinated epidemiological surveillance systems. In addition, under reporting of cases and the presence of other diseases considered to be of higher priority may have overshadowed the problem of food-borne diseases including Salmonellosis. Therefore, the aims of the current study were: To isolate and identify Salmonella from dairy cows

feces, personnel hand, equipment and contaminated floor sample and to evaluate the antibiogram pattern of the isolates to commonly used antibiotic agents from selected dairy farms in Modjo and Adama towns. 

Study Area – The study was conducted in Modjo and Adama towns from February 2019 to June 2019. Modjo is the administrative center of Lome district, located in the East Shewa Zone of the Oromia Region, Ethiopia. It is located at 66 Km Southeast of Addis Ababa and lies at latitude 8°35N and longitude 39°7E at an altitude of 1790 meters above sea level. The area gain rainfall twice a year those known as long and short rainy season. The main rainy season extends from June to September. The average annual rainfall, temperature, and mean relative humidity are: 776 mm, 19.4ºC and 59.9% respectively (CSA, 2005).Adama  town, East Shoa, Ethiopia, which  is  located  about  100  km  south  east  of  Addis  Ababa  at  an altitude  of  1650  meter  above  sea  level.  Its annual temperature ranges from 13.9oC - 29oC. The mean annual rainfall of the area is 1024 mm.  The livestock population of the area in 2004/2002 estimated to be 70,622 cattle, 36,142 sheep, 42,968 goats and 2,193 equines (CSA, 2005). 

Study Population - The study population is lactating dairy cows in Modjo and Adama towns and the study animals were apparently healthy dairy cows in small- and large-scale dairy farms located in and around Modjo and Adama towns. The farms were selected by using simple random sampling strategies based on data obtained from both areas livestock and fishery resource development. In this study, the majority of farms found in the study areas were small scale having herd sizes not more than six cows. All of the available lactating cows present in each farm were sampled. According to personal observation, the hygienic status of the cows and their environment was more or less good even though some animals were reared under poor hygienic condition plus in a manner mixed with other activities of the households. Farm equipment used in the milking and storage of milk and personnels (milkers) were also part of the study.

Study Design - A cross-sectional study was carried out to isolate, identify and detect antimicrobial susceptibility profile of the Salmonella from dairy farms. Sampling days were randomly assigned and each farm was visited only once during the study period. Types of sample collected include feces, bulk tank milk, milkers hand swab, contamination of floor. 

Sample Collection and Transportation - Samples from dairy cows (faces), hand swab of personnel working in the farms (milkers), from equipment and from contaminated environment (contaminated floor sample) were aseptically collected from the selected dairy farms. Samples from dairy cows were collected from apparently health lactating cows. Fresh fecal samples were collected directly from the rectum of healthy lactating dairy cows using disposable gloves into sterile plastic bags. Pooled milkers hand swab is collected before the beginning of milking process by using a sterile cotton swab. Floor samples were collected from bedding of the animal house and from the environment and equipment which the cows feed using a sterile a sterile cotton swab and Samples were properly coded based on collection date, sample source and sample type. Source of sample was classified as animal, personnel and equipment. Types of samples collected in quantity were faces (89), pooled milkers hand swab (9), bulk tank milk (10) and floor sample (9). Then samples were immediately transported under cold condition (ice box) to the Microbiology Laboratory of College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoft. Upon arrival, samples were processed separately by pre-enriching in pre-enrichment media or were stored overnight in a refrigerator at +4oC until examined the next day. Then further processes were followed after samples were incubated for 24 hrs.

Isolation and Identification of Salmonella 

Pre-enrichment and selective enrichment - The feces samples were pre-enriched  in  appropriate  amount  of  buffered  peptone  water  in (1:9)  ratio  and  incubated  at  37°C  for  24  hrs.  Rappaport-Vassiliadis  medium  (RV)  broth and Selenite (SB) broth  were  used  for selective  enrichment  of  the  samples. About  0.5 and  ml  of  the  pre-enriched  sample  was transferred into  a  tube  containing 10  ml  of  Rappaport-Vassiliadis medium  (RV broth)  and incubated at 42°C for 24 hours. Another 1.0 ml of the pre-enriched broth was transferred into a tube containing 10ml of Selenite broth and incubated at 37°C for 24 hours.  

Plating out and identification – Xylose lysine deso-xycholate  (XLD) agar plate was used for plating out and identification. A loop full of inoculums from each RV and Selenite broth cultures were plated onto XLD plate and incubated at 37oC for 24 hours. After  incubation,  the  plate was examined  for  the  presence  of  typical  and suspect  colonies. Typical colonies of Salmonella grown on XLD-agar have a black center and a lightly transparent zone of reddish color due to the color change of the media while  H2S  negative  variants  grown  on  XLD  agar  are  pink  with  a  darker  pink center. Lactose-positive  Salmonella  grown  on  XLD  agar  are  yellow  with  or  without blackening. Five  typical  or  suspected  colonies  were selected  from  the  selective  plating  media,  streaked  onto  the  surface  of  pre-dried  nutrient agar  plates  and  incubated  at  37oC  for  24hrs. All suspected Salmonella colonies were picked from the nutrient agar and inoculated into the following biochemical tubes for identification: triple sugar iron (OXOID CM0277, England) agar, Simmons citrate agar (HIMEDIA M099, India), Indole  tests  (Becton  Dickinson, USA), Methyl red-Voges-Proskauer (HIMEDIA M070, India) broth and then incubated for 24 to 48 hrs at 37oC

Biochemical Confirmation of Salmonella Isolates - All suspected Salmonella isolates were subjected to the following biochemical tests for confirmation: Triple Sugar Iron (TSI) test, Indole test, Citrate utilization test, Methyl red test, and Vogues Proskauer (VP) test. Colonies producing red slant (alkaline), with yellow butt (acid) on TSIA with blackening due to hydrogen sulphide (H2S) production and e (gas production) in butt, negative for Indole test, positive for Methyl red test (red broth culture), positive for citrate utilization (deep blue  slant),  and  negative  for  VP test  were considered  to  be  Salmonella  positive  (Quinn  et al.,  2004). Presumptive Salmonella isolates that were found fulfilled the Salmonella characteristics on all biochemical tests indicated above were transferred and cultured on Nutrient Agar (NA) for antimicrobial sensitivity and motility tests. 

Antimicrobial Susceptibility Testing - The antimicrobial susceptibility testing of the isolates was performed with Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute of U.S.A (CLSI, USA) and Kirby Bauer Disk Diffusion Susceptibility Test Protocol on  Muller  Hinton  agar  medium.  From each biochemically confirmed isolate, loop full of well grown colonies on nutrient agar were transferred with sterile loop into sterile tubes containing 5ml of normal saline solution (0.85% NaCl). The inoculated colonies mixed well with saline solution by vortex until smooth suspension was formed. Saline solution (if suspension more turbid) or colonies (if suspension less turgid) were added to the suspension until it achieved to the 0.5 McFarland turbidity standards. Then sterile cotton swab was dipped into the suspension and the bacteria were swabbed uniformly over the entire surface of Muller Hilton Agar plate. The plates were being held at room temperature for 3 minutes in bio-safety cabinet to allow drying. Ten antimicrobial disks with known concentration of antimicrobial were placed on the Muller Hinton Agar plate; nine of them in circular pattern and one at the center and the plates were incubated for 22 hrs at 37ºC.

Each isolate was tested for a series of eleven antimicrobials: Tetracycline (TE) (30µg), Cefoxitin (FOX) (30µg), Cefuroxime (30µg), Ciprofloxacin (CIP) (5µg), Gentamycin      (CN) (10µg), Nitro-furantoine (NIT) (300μg), Nalidixic acid (NA) (30µg), Streptomycin (S) (10µg), Erythromycine (ERY) (15) and Methicillin (ME) (5). The diameters of clear zone of inhibition produced by diffused antimicrobial on lawn inoculated bacterial colonies were measured to the nearest mm using caliper. All eleven zone of inhibition against eleven antimicrobial agents for each isolate were recorded and compared with standards and interpreted as resistant, intermediate, or susceptible according to published interpretive chart (CLSI, 2013). 

Data Management and Analysis - The raw data generated from the study were arranged, organized, coded and  entered to Excel spread sheet (Microsoft® office excel 2007). Then the data was analyzed using SPSS version 20 through descriptive analysis with chi-square statistics. The results of analyses were mostly described in proportion. Proportion were estimated as the  numbers of samples detected positive to  Salmonella  from the total sample tested as well as the numbers of antimicrobial resistant isolate to the detected positive isolate.

Growth on Solid Media - On nutrient agar Salmonella colonies were moderately large (2-4 mm), circular with smooth surface and grayish- white in color after 24 hours at 37ºC (Fig 1). Growth on deoxycholate agar showed slight opaque dome- shaped colonies measured (2-4 mm) with central black spots (indicated production of hydrogen sulfide) surrounded by a zone of clearance after 48 hours at 37ºC (Fig 2). On triple sugar iron agar Salmonella colonies produced hydrogen sulfide which was indicated by black discoloration, gas production causes bubbles in the agar, and pH change was indicated by production of red color in the slant (Fig 3).

Proportion of Bacteria Isolation - Salmonella was isolated from 10/117 (8.5%) of the total samples. Out of the 10 Salmonella isolates, 8 (80%), 1 (10%), (and 1 (10%) were from feces, bulk tank milk, and floor sample, respectively (Table 1). From a total of 89 cows feces examined, 8.98% (8/89), 10 bulk tank milk samples, 10% (1/10), and out of 9 contaminated floor sample, 11.1% (1/9) were confirmed positive for Salmonella from both cities, Adama and Modjo. 

Fig 1: Growth of Salmonella on Nutrient Agar.

Fig 2: Growth of Salmonella on Xylose Lysine Desoxychocolate.

Fig 3: Growth of Salmonella on Triple Sugar Iron Agar (TSI).

Table 1: Frequency of Salmonella isolated from different sample types in the study area.

There is no statistically significant difference between isolates derived from different sample types of the studied dairy farms (χ2=0.966, p=0.809). From a total of 10 (8.5%) isolates, nine (90%) were from Adama and 1 (10%) was from Modjo. There is statistically significant difference between isolates derived from Adama and Modjo of studied sites (χ2=6.012, p=0.014) (Table 1). 

Antibiotic Susceptibility Testing - All the 10 isolates were tested against ten commonly used antimicrobials (Table 2). All isolates were resistant at least to one or more antimicrobials. Nine of the 10 isolates were resistant to two or more antimicrobials. The antibiotic susceptibility profiles of the isolates showed that the isolates were 60% resistant to all of the antibiotics which are streptomycin, methicillin and nalidixic acid. Gentamycin was the most effective antibiotic. Ninety percent (9/10) of the isolates were found to be susceptible to gentamycin (Fig 4).

Multi-Drug Resistance Frequency Distribution - Among  10  resistant  isolates, 8 (80%)  were  resistant  to  two  or  more  antimicrobials  (multidrug resistance (MDR)). The large proportion of multi-drug resistant isolates 6 (60%) were resistant to four to seven  different  antimicrobials  while  the  other  two  resistant  isolates  were resistant  to  a single antimicrobial.  2 (6.67%), 5 (16.67%), 4 (13.33%), and 7 (23.33%) were tetra-resistant, quadra-resistant, 

hexa-resistant, hepta-resistant, respectively with 11 different resistance patterns. Among 8 MDR isolates, 6 (75%) from feces, 1 (12.5%) bulk tank milk, and 1 (12.5%) floor sample isolates (Table 3). 

Sensitivity to antimicrobial agents - Sensitivity test to the ten Salmonella isolates against 10 antibacterial agents was carried out. 60% of Salmonella isolates were found resistant to streptomycin, methicillin and nalidixic acid, and 90% of the isolates sensitive to gentamycin (Fig 5). 

Salmonellosis is the most common food borne disease in both developing and developed countries, although incidence rates vary according to the country (Stevens et al., 2006). The fecal wastes from infected animals and humans are important sources of bacterial contamination of the environment and the food chain (Ponce et al., 2008). 

Salmonella infection in dairy cattle persists to be a major problem worldwide. Considerable economic losses were manifested through mortality and poor growth of infected animals as well as the risk of transmission to humans either through food chain or direct animal contact (Alam et al., 2017). Hence, detection of animals contacting humans and equipment are essential to control Salmonella on farm and its spread to the public (Rotimi et al., 2008).

Table 2: Antibiotic susceptibility profiles of Salmonella isolates in dairy farms.

This cross-sectional study showed that Salmonella was isolated (8.5%) from dairy farms in Adama (12.9%) and Modjo (1.8%) with variable isolation rate. This result is significantly high to be a potential source of food borne Salmonellosis in humans. High proportion (80%) of Salmonella isolates were resistant to two or more of the antimicrobials that are commonly used in the veterinary and public health set up. This may pose difficulties in the treatment of human clinical cases and other bacterial diseases. Based on the above conclusion the following recommendations are forwarded: Hygiene status of the dairy farms should be improved to minimize cross contamination of Salmonella from milking containers and cattles environment, Since  Salmonella  is  resistant  to  most  common  drugs,  attention  should  be  taken  in selecting  antimicrobials  in  treating  Salmonella  infection  both  in  animals  and  human being  based on antimicrobial susceptibility  test, Further study ought to be conducted to identify the source of contamination, and Molecular characterization of the isolates with emphasis on resistant strains is also necessary to identify mechanisms of antibiotic resistance.

The author is thankful to his co-authors who encouraged and supported to the successful research.

The author declares that there is no conflict of interest about the publication of the article.