Outbreak Investigation in Chicken Farms at Bishoftu and Ejere Towns, Central Ethiopia
Umer AA and Mezgebu E
Published on: 2023-08-28
Abstract
Salmonella species and Escherichia coli (E. coli) are cause salmonellosis and colibacillosis in chickens respectively. Poultry outbreak cases occurred in Bishoftu and Ejere town from June to July 2023. This case is aimed to assess E. coli and Salmonella isolates and antimicrobial susceptibility test (AST) from poultry farms. During the investigation, chickens showed clinical signs of sudden death, loss of appetite, ruffled feathers, droopiness, sneezing, yellow diarrhea, huddling of chicks, dehydration, and morbidity were observed. A post-mortem examination was conducted on the chicks and alterations and gross lesions including livers were swollen and the Ischemic lobe, soft, congested, and revealed bronze discoloration and hyperemic spleen was observed. A total of 8 pooled tissue samples: four from each (Bishoftu and Ejere) towns were collected. Bacteriological and Omni log identification procedures were used to isolate and confirm the bacterium. Following that, antibiotic susceptibility tests were performed on confirmed isolates. Escherichia coli and Salmonella spp have been isolated from kidney, spleen, liver and intestine samples with each pathogen having a 75% occurrence. In Bishoftu and Ejere the frequency of Salmonella spp was 0 % (0/4), and 75% (3/4) respectively, while that of E. coli was 25% (1/4) and 50% (2/4) respectively. AST to Salmonella spp showed resistance towards Streptomycin (S; 10 μg), whereas it was susceptible to antimicrobials including Tetracycline (TE; 30 μg), Norfloxacin (NOR; 10 μg), Amoxicillin clavulanate (AMC; 30 μg), Ceftriaxone (CRO; 300 μg), Ampicillin (AMP; 10 μg), Meropenem (MEM; 10μg), Sulfonamides (S3; 300), Ciprofloxacin (CIP 5; μg) respectively. The resistance was recorded towards both pathogens; Tetracycline (TE; 30 μg) for E. coli and Streptomycin (S; 10 μg) for Salmonella Gallinarum. This outbreak-finding would certainly assist chicken farm owners to select appropriate antibiotics against colibacillosis and salmonellosis diseases. This work also demonstrated a high prevalence of Salmonella spp. and E. coli in Ejere chicken farms. This could be due to less biosecurity implementation, less farm management, and inappropriate use of antimicrobial agents in these farms. Strong Biosecurity measures and hygiene should be very essential in chicken farms for prevention and control of these diseases
Keywords
Outbreak; E. coli; Salmonella; AST; Poultry; Bishoftu; EjereIntroduction
Poultry production in Ethiopia is rising, but it still confronts several problems, including infectious disease outbreaks of avian salmonellosis and colibacillosis [1]. Antimicrobials are widely used in poultry for the treatment and prevention of infectious disease, and excessive use and misuse of antimicrobials has been linked to rising rates of antimicrobial resistance among pathogens isolated from poultry [2]. Escherichia coli (E. coli) is a bacteria that exist in the intestine of animals and humans as commensal, however diseases can be caused in poultry from pathogenic strains [3]. E. coli and Salmonella are gram-negative, rod-shaped, facultatively anaerobic, non-spore forming bacteria and belong to the family of Enterobacteriaceae [4]. Poultry salmonellosis can be caused by invasion and intestinal colonization by Salmonella serovars causing in septicemia, enteritis and mortality.
Certain serovars of Salmonella, such as Salmonella Typhimurium and Salmonella Enteritidis, may persist in the digestive tract of chickens. Avian colibacillosis is one of the most serious infectious diseases caused by E. coli strains. It is a major economic burden on the poultry industry, resulting in high morbidity, mortality rates, and high treatment and prevention costs [5].Most of them are motile; catalase positive; oxidase positive and urease negative. Pullorum disease (PD) and Fowl typhoid (FT) are a septicemic disease caused by bacterium Salmonella Pullorum (S. Pullorum) and Salmonella Gallinarum (S.Gallinarum), which is host-specific in chickens. These strains are not zoonotic in nature, as they do not infect other hosts. PD causes severe mortality in poultry birds, resulting in massive economic losses. These birds serve as a reservoir of disease transmission to other uninfected birds [6]. Antimicrobial resistance (AMR) in chickens is one of the most common challenges in developing countries. This is due to the widespread use of antimicrobial agents in feed additives and in the prophylaxis of infections. Due to the high prevalence of antimicrobial agents, some organisms are able to resist most or all of the antimicrobial agents [7].
Case Study and Investigation
An outbreak investigation was carried out following the report from Bishoftu and Ejere poultry farms from June to July 2023. From the 8,166 total chicken population at risk, 94 mortality at 41 weeks of age were recorded in the farm and 10 live suspected poultry were presented for investigation. It was reported that chickens were died after presenting clinical symptoms. During the outbreak investigation clinical and postmortem examination, and Laboratory analysis were conducted. Eight to ten live chicken samples from each farm were submitted to Animal Health Institute, Sebeta.
During postmortem examination, a lesion of pooled four types of tissue samples involving the liver, kidney, spleen, and intestine were collected, and submitted to the bacteriology laboratory and analyzed for confirmation. Finally, the cases were confirmed as E. coli and Salmonella after bacterial culture, Biochemical test, and Omni log bacterial test identification were conducted; and most of the chickens were positive for three (3) E. coli and three (3) Salmonella species.
Outbreak Area
The case was reported from Bishoftu and Ejere town, Oromia regional state, central Ethiopia. In both towns urban agricultural practices are present in the area and also Bishoftu town is known by commercial poultry farm production.
Bishoftu town is situated in the eastern Shewa Zone of the Oromia Region. It is located approximately 45km southeast of Addis Ababa, the capital of the region. Bishoftu is found at a latitude of 9°N and a longitude of 40°E at an elevation of 1850 meters above sea level. According to the national meteorological agency [8], the average annual rainfall in Bishoftu is 866 mm, 84% of which falls during the long rainy season from June to September. Annual minimum and maximum temperatures in the town are 11 degrees Celsius and 29 degrees Celsius respectively. In this town, there are 43138 poultry, 30887 cattle, 9322 horses, 9294 sheep, and 4753 goats that are raised. Ejere town is situated in the West Shewa Zone of the Oromia Region, west of Addis Abeba. Its latitude and longitude are 9°2′N and 38°24′E, respectively, and its elevation is about 2360 meters above sea level.
Diagnostic Approach
Clinical Signs and Post Mortem Lesions Examination
The clinical signs observed were in appetence, sudden death, weakness, ruffled feathers, yellowish diarrhea and illness. The diseased chicken was conducted for post-mortem examinations to observe alterations and gross lesions. From the organs examined livers (Figure 1) were invariably enlarged and the Ischemic lobe, congested and discoloration.
Figure 1: (A): Liver Lobe Lesion And (B): Greyish To Whitish Necrosis With Spots Are Found On The Liver.
Sample Collection
During postmortem examination, four types of tissues including; Liver, kidney, spleen and intestine having lesions were collected aseptically. Culturing on selective media, examination of colony characteristics, and various biochemical tests were carried out to identify Salmonella spp and E. coli (Figure 2).
Figure 2: Tissue Samples Collected During Post Mortem Examination.
Laboratory Analysis
Escherichia coli and Salmonella Gallinarum were identified by bacterial culture, biochemical test, and Omni log bacterial identification test.
Bacterial Culture and Biochemical Test
Isolation of E. coli was carried out by suspending 1 g of tissue sample into 9 ml of Tryptone Soya Broth (oxoid, UK). Samples were incubated overnight at 37°C. After pre-enrichment, a loop full broth was streaked onto Eosin Methylene Blue (EMB; oxoid, UK) agar and incubated for 24 hours at 37°C. Then, colonies that displayed a metallic sheen were suspected to be E. coli. From EMB a pure single colony was inoculated onto nutrient agar (oxoid, UK), and biochemical tests were performed to confirm the E. coli using Indole, Methyl red, Voges-Proskauer, and Simmon’s Citrate tests. Then, the E. coli suspected bacterium was subculture onto biology universal growth (BUG) agar and confirmed by Omni log. Then, the Omni log confirmed E. coli were subjected to AST [9].
Isolation and identification of Salmonella were conducted according to [10], 1 g of tissue was suspended into 10 ml of buffered peptone water (BPW) (oxoid, UK) and incubated overnight at 37°C and 0.1 mL of bacteria with BPW was inoculated into Rappaport Vassiliadis Soya (RVS)
Broth (Oxoid, UK) at 41.5?C for 24 hours for selective enrichment. Following that, the RVS mixture was streaked on Xylose-lysine-desoxycholate (XLD) agar (Oxoid, UK) and incubated aerobically overnight at 37 ?C. After 24 hrs, the plate was examined for the presence of suspected Salmonella spp. The suspected colonies were inoculated into nutrient agar and subjected to biochemical tests including Triple sugar iron agar (TSI), Urease, Indole, Methyl red, Voges-Proskauer, and Simon’s citrate test. Biochemically suspected Salmonella was being inoculated into BUG and confirmed by Omni log. Then, the Omni log confirmed Salmonella were subjected to AST [11].
Antimicrobial Susceptibility Test (AST)
The Clinical Laboratory Standard Institute (CLIS) is used for antimicrobial disk diffusion test to assess the antimicrobial susceptibility of each isolates. The commercial antimicrobial disc from (Oxoid, UK) listed below was used: Tetracycline (TE; 30 μg), Norfloxacin (NOR; 10 μg), Amoxicillin-clavulanate, Ceftriaxone (CRO; 300 μg) Ampicillin (AMP; 10 μg), Meropenem (MEM; 10μg) Sulfonamides (S3; 300) Streptomycin (S; 10 μg), Ciprofloxacin (CIP 5; μg). All chosen antimicrobials are frequently used to treat Salmonella and E. coli infections according to [12]. A 0.5 McFarland bacterial suspension was prepared and streaked on the Muller hinton agar surface. A total of nine antimicrobial discs were placed onto each agar plate using a disc dispenser. For 16 to 18 hours, the plate was incubated at 37°C. Using a caliper, the zones of inhibition were measured in millimeters, and the measurements were recorded. The zone of inhibition diameter breakpoints and interpretive categories were noted as susceptible, intermediate, or resistant to determine the antimicrobial sensitivity profiles of the isolates [13].
Results
Isolation of Salmonella and E. coli
From a total of 8 pooled samples collected from two chicken farms, 8 samples were examined for E.coli and Salmonella, and 3 (75%) isolates were identified for each pathogen. The isolation of E.coli and Salmonella in the pooled of different tissue samples including liver, intestine, kidney and spleen were examined as indicated in (Table 1).
Salmonella Gallinarum causing infections 75% and E. coli 50% in Ejere chicken farm. However in Bishoftu 0% Salmonella spp and E. coli 25% in causing infections. The overall results were equal rate infection of both pathogen in farms. Antimicrobials susceptible test of Salmonella spp was displayed resistance towards Streptomycin (S; 10 μg), whereas the following antimicrobials were susceptible Tetracycline (TE; 30 μg), Norfloxacin (NOR; 10 μg), Amoxicillin clavulanate (AMC; 30 μg), Ceftriaxone (CRO; 300 μg), Ampicillin (AMP; 10 μg), Meropenem (MEM; 10μg), Sulfonamides (S3; 300), Ciprofloxacin (CIP 5; μg) respectively. The resistance were recorded towards both pathogens; Tetracycline (TE; 30 μg) for E.coli and Streptomycin (S; 10 μg) for Salmonella Gallinarum.
Table 1: The Occurrence of E.coli and Salmonella in Bishoftu and Ejere Poultry Farm.
Poultry Farm |
Pathogen Identified |
Organ |
Pooled Samples |
% of Detection |
Bishoftu |
Salmonella Gallinarum |
All |
0 /4 |
0% |
E. coli |
Kidney |
¼ |
25% |
|
Ejere |
Salmonella Gallinarium |
Spleen, Liver and kidney |
¾ |
75% |
E. coli |
Liver and intestine |
02-Apr |
50% |
Figure 3: Salmonella Identification on XLD (Bright Pink Color Colony) and Urea (Negative Yellow Color).
Figure 4: Escherichia Coli Identification on XLD (Yellow Colony) and EMB (Metallic Sheen Colony).
Figure 5: Omni Log Detection Report.
Table 2: Antimicrobial Susceptibility Test for E.coli and Salmonella Spp.
Antibiotics |
Isolates |
Resistance |
Intermediate |
Sensitive |
Tetracycline(TE; 30 μg) |
6 |
1 |
5 |
|
Ciprofloxacin (CIP 5; μg) |
6 |
6 |
||
Streptomycin (S; 10 μg) |
6 |
1 |
5 |
|
Sulfonamides(S3;300) |
6 |
6 |
||
Meropenem(MEM; 10μg) |
6 |
6 |
||
ampicillin (AMP; 10 μg) |
6 |
6 |
||
Ceftriaxone(CRO; 300 μg) |
6 |
6 |
||
Amoxicillin-clavulanate (AMC;30 μg) |
6 |
6 |
||
Norfloxacin(NOR;10 μg), |
6 |
6 |
Discussion
In poultry, Salmonella and E. coli are the major microorganisms related with bacterial diseases. In the present outbreak, the findings of the Clinical symptom and gross lesions seem to be consistent with the results of [14]. In the case here reported, Salmonella was isolated from Ejere from different organs which is in agreement with previous reports [15]. However, none was isolated from the Bishoftu, which demonstrates that the poultry was not shedding this bacterium at the moment and was not a carrier in Bishoftu.
The E.coli was isolated from Ejere and Bishoftu, which indicates that the poultry was shedding this bacterium at the moment. This possibility may be important since this spread of pathogens in different locations [16]. The results of our investigation are consistent with those of a Malaysian study, which found a high prevalence rate of E. coli (60%) [5]. Antimicrobial susceptibility tests were conducted using disc diffusion methods to evaluate the susceptibility of selected antimicrobials to selected Salmonella spp. and E. coli isolates. Both of these bacteria were resistant to some of these antimicrobials and, Salmonella spp displayed resistance to Streptomycin (S; 10 μg), whereas the E. coli to Tetracycline (TE; 30 μg) which is in agreement with previous studies [5]. Due to the widespread use of antimicrobials as feed additives and a prophylactic measure for infectious diseases, AMR in chickens is an issue in developing countries [11].
Conclusion and Recommendations
Salmonellosis and colibacillosis pose a high risk and the most prevalent infections that affect poultry worldwide. It causes significant economic losses and makes it exceedingly challenging to control the disease. Antimicrobials were administered either as a form of treatment or as growth promoters, both of which increase the risk of bacterial resistance to antimicrobials developing. These organisms are known to cause major challenges with chicken health that result in mortality, decreased productivity, and higher expenses for disease prevention and treatment. In summary, this finding suggested to stop the spread of infections, control measures and rigorous monitoring must be put in place, and biosecurity precautions must be promoted. S. Gallinarum and S. Pullorum were host specific and cause infections in poultry birds.
Consuming contaminated feed and water is the main cause of E. coli and Salmonella infection. Furthermore, a serious problem for poultry still exists in countries where control measures are inefficient and where it was found that farms were the main locations of the majority of outbreaks of Salmonella and E. coli. Spraying and fumigating the entire farm with formaldehyde to disinfect it is an essential stage in the disease-control strategy. Salmonella and E. coli are challenging because they may persist in the environment. Salmonellosis and Colibacillosis can be controlled from farms by closely adhering to biosecurity measures including disinfection and basic hygiene practices. In order to control the bacterial infections, prompt treatment is necessary.
Conflicts of Interest
No conflict of interest is declared
References
- Serbessa TA, Geleta YG, Terfa IO. Review on Diseases and Health Management of Poultry and Swine. Int J Avian & Wildlife Biol. 2023; 7: 27-38.
- Ibrahim S, Hoong LW, Siong YL, Mustapha Z, Aklilu E, Mohamad M, et al. Prevalence of Antimicrobial Resistance (AMR) Salmonella spp.and Escherichia coli Isolated from Broilers in the East Coast Malaysia. 2020; 1-21.
- Tammisettty, Reddy AD, Karuna Sree E, Subbaiah KV, Raju GS, Reddy RVSK. Salmonellosis in Poultry?: A Case Report. Int J Curr Microbiol App Sci. 2018; 7: 2347-2349.
- Samad A, Hamza M, Muazzam A, Ahmer A, Tariq S, Shahid MJ, et al. Overview Of Bacterial Diseases In Poultry And Policies To Control Disease And Antibiotic Resistance. J Multidisiplin Ilmu. 2022; 1: 1-7.
- Ibrahim S, Hoong LW, Siong YL, Mustapha Z, Aklilu E, Mohamad M, et al. Prevalence of Antimicrobial Resistance ( AMR ) Salmonella spp . and Escherichia coli Isolated from Broilers in the East Coast of Peninsular Malaysia. Antibiotics. 2021; 10: 1-11.
- Wibisono FM, Wibisono FJ, Effendi ME, Plumeriastuti H, Hidayatullah AR, Hartadi EB, et al. A Review of Salmonellosis on Poultry Farms?: Public Health Importance. 2020; 11: 481-486.
- Varga C, Guerin MT, Brash Ml, Slavic D, Boerlin P, Susta L. Antimicrobial Resistance In Fecal Escherichia Coli and Salmonella Enterica Isolates?: A Two- Year Prospective Study Of Small Poultry Flocks In Ontario, Canada. BMC Veterinary Research. 2019; 15: 1-10.
- Ababa A. National Meteorological Agency. REVISED METEOROLOGICAL STATION NETWORK MASTER PLAN. Revised Meteorological Station Network Master Plan Preparation technical committee. 2021.
- Geletu US, Usmael MA, Ibrahim AM. Isolation, Identification, and Susceptibility Profile of E. coli, Salmonella, and S. aureus in Dairy Farm and Their Public Health Implication in Central Ethiopia. Vet Med I. 2022; 1-13.
- ISO 6579-1. INTERNATIONAL STANDARD. Horizontal method for the detection, enumeration and serotyping. 2017.
- Abdi RD, Mengstie F, Beyi AF, Beyene T, Waktole H, Mammo B, et al. Determination Of The Sources And Antimicrobial Resistance Patterns Of Salmonella Isolated From The Poultry Industry In Southern Ethiopia. BMC Infect Dis. 2017; 17: 1-12.
- Testing. M100-S25 Performance Standards for Antimicrobial. January. 2015.
- Hombach M, Maurer FP, Pfiffner T, Böttger EC, Furrer R. Standardization of Operator-Dependent Variables Affecting Precision and Accuracy of the Disk Diffusion Method for Antibiotic Susceptibility Testing. J Clin Microbiol. 2015; 53: 3864-3869.
- Petros H, Masebo NT, Meaza M. Major Causes of Chicken Mortality and Gross Pathological Study on Commercial Poultry Farms in Wolaita Sodo Town, Southern Ethiopia. 2018; 12: 153-161.
- Sarba EJ, Kudama K, Dandecha M, Megersa L, Borena BM, Gebremdhin EJ. Prevalence, organ distribution and antimicrobial susceptibility profile of Salmonella isolated from chickens purchased from markets in selected districts of West Shoa, Ethiopia. Ethiop Vet J. 2020; 24: 73-89.
- Abda S, Haile T, Abera M. Isolation, Identification Antimicrobial Susceptibility and Associated Risk Factors of Salmonella in Semi-Intensive Poultry Farms Of Kafa Zone, Southwest Ethiopia. Vet Anim Sci. 2021; 14.