Study Area
Study was conduct in Jimma town which is situated in south western Ethiopia. Jimma town is located at latitude of 07o41”N and longitude of 36050” E, and at an elevation average of 1780m above sea level [14].
Study Design
Cross-sectional study design was conduct from January 2018 to September 2018 on egg and cloacae swab of chicken. The number of eggs sample were estimated based on previous reports using Thrusfield formula [15].
Accordingly, 4.69% [13] expected prevalence was taken with 5% desired absolute precision and 95% confidence interval. Samples size was separately calculated for eggs sampled from markets and farms.
Calculated sample size was ≈69 for each. This was increased by 20% and 83 eggs were sampled from each (market and farm). From 166 total eggs, 83 eggs contents and 83 eggshells of market eggs samples and 83 eggs content and 83 eggshell of farm egg samples were analyzed separately. Similarly, 83 cloacae swab samples were collected from chicken those laid sample of egg at farm. Overall, 415 samples were tested for salmonella detection.
Samples from poultry farms were collected using proportional allocation sampling method and allocated samples were collected randomly. Samples of egg from markets were randomly collected. Structured questionnaire was administered to egg collectors and egg sellers at the markets to assess factors favoring contamination of egg with salmonella.
Sample Collection and Transportation
Sample of egg from farms were collected as soon as egg is laid using sterile glove. Cloacae swabs were collected according to [16] and swabs were placed in sterile tube containing10ml of Buffered Peptone Water (BPW). Samples of egg from markets were collected using sterile glove. Each sample was coded, packaged separately in an ice box and transported to analyze laboratory.
Sample Processing
Sterile cotton tipped swab was soaked in BPW and external egg was rubbed. Swab was inoculated in 10ml of BPW. Eggshell was washed and immersed in 70% alcohol. Eggs were cracked and 25 gram of egg content was added into flask. 225 ml of trypticase soy broth (TSB) was added on the egg content in the same flask, mixed and incubated according to [18].
Salmonella Isolation and Identification
Salmonella isolation was performed as recommended by [17]. Briefly, 1ml of BPW mixture of eggshells, cloacae swabs and 1ml from incubated TSB with egg content mixture were transferred to 10 ml Selenite cysteine broth (SCB) and incubated. A loop full from incubated SCB was streaked on XLD and BGA and incubated. Plate was examined for the presence of Salmonella [19].
Salmonella suspected isolates on BGA and XLD were tested via biochemical test according to [20, 21]. Isolates producing an alkaline slant with acid butt on TSI and H2S production or no H2S production, urea hydrolysis negative and indole negative, citrate utilization positive, decarboxylate lysine positive and motile were assumed as salmonella species.
Salmonella isolates confirmed by biochemical test were taken to Biolog OmniLog test. This was by growing salmonella isolates on Biolog Universal Growth Agar. Cell suspensions was made and pipette into 96 well of Biolog Plates and incubated [22]. The incubated microplates were inserted in to Biolog OmniLog reader and analyzed. Result was read from computer software [23].
Antibiogram of Salmonella Isolates
Phenotipically confirmed Salmonella isolates were subjected to twelve (12) antimicrobial discs by agar diffusion method [24]. Culture of isolates were compared with 0.5 McFarland turbidity standards and swabbed on Mueller Hinton Agar [4]. Antimicrobial discs were placed on Mueller Hinton Agar and incubated. For each antimicrobial, inhibition zone was measured.
Associated Risk Factors
Structured questionnaire was pretested and administered to interviewee (farm managers, egg collectors and egg sellers at the market) to assess potential factors favoring contamination of egg with salmonella species. The structured questionnaire survey at farm was includes; number of chicken in each farm, Chicken Keeping system, availability of disinfection bath at the entrance of the farm, eggs collection methods, feeding methods, while farm workers washing their hand after use of toilet, use of protective cloth, cleaning of stained/dirty/ eggs, entrance of other people in to farm, washing egg collection material/container, separating of cracked eggs from undamaged eggs, treatment of poultry with antibiotic medication, mixing of chicken excreta with fodder and eggs. The structured questionnaire survey at market was include; maximum number of days the unsold egg stays at market, using storage/frigid for unsold egg, cleanliness of egg containers, mixing eggs bought from different farmers, and separating cracked eggs.
Data Collection, Management and Analysis
Data collected from laboratory investigation and questionnaire survey were stored. In univariable logistic regression, all independent variables with P-value <0.25 were taken to multivariable logistic regression. Independent variables with P< 0.05 in multivariable logistic regression were considered as significant.