African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5181

Full Length Research Paper

Isolation and characterization of bacteria associated with yolk sac infection (Omphalitis) in chicken from three hatcheries in Bishoftu, Ethiopia

Kasech Melese
  • Kasech Melese
  • Debre-zeit Agricultural Research Centre, Institute of Agricultural Research, Bishoftu/Debre-zeit, Ethiopia.
  • Google Scholar
Wondmeneh Esatu
  • Wondmeneh Esatu
  • Debre-zeit Agricultural Research Centre, Institute of Agricultural Research, Bishoftu/Debre-zeit, Ethiopia.
  • Google Scholar
Takele Abayneh
  • Takele Abayneh
  • National Veterinary Institute, Bishoftu/Debre-zeit, Ethiopia.
  • Google Scholar

  •  Received: 01 November 2016
  •  Accepted: 25 October 2017
  •  Published: 21 November 2017


A study was conducted from November 2014 to June 2015 at three hatcheries designated as A, D and E in Bishoftu Town, Ethiopia, to isolate and identify bacteria associated with yolk sac infection and to determine antimicrobial susceptibility profile of the predominant isolates. A total of 385 Lohmann and Koekoek breed, 1 to 7 days old chicks suffering from yolk sac infection were examined from three different hatcheries of which 96.1% (N=370) of them were showing unabsorbed yolk sac. All the chicks were necropsied and yolk sac samples were collected for isolation and identification of bacteria followed by testing of the isolates for their susceptibility to 11 antimicrobial agents using disc diffusion method. A total of 323 bacterial isolates were identified, of these Escherichia coli were the most common bacteria (N=116; 35.91%) isolated followed by Salmonella species (N=111; 34.36%) and Staphylococcus aureus (N=96; 29.72%). Significant difference (P<0.001) was noted among the hatcheries on the frequency of isolation of the predominant bacteria species from yolk sac samples with the highest rate of isolation being in hatchery A. All the tested predominant bacterial isolates showed higher susceptibility to Gentamicin, Chloramphenicol, Amikacin (except S. aureus) and Kanamycin but were resistant to Ciprofloxacin, (except S. aureus), Penicillin G, Tetracycline, Sulfamethoxazole  and Amoxicillin (except S. aureus). The existence of multi-drug resistant bacteria isolates associated with yolk sac infection suggests that more emphasis should be given towards preventing omphalitis in chicks through improvements of sanitary measures at hatcheries than to use antimicrobials to control infections.


Key words:  Bacteria, chicken, yolk sac infection, antimicrobial sensitivity, Ethiopia.


The rapid expansion of poultry industry has presented many poultry diseases. Yolk Sac Infection (YSI) also known as mushy chick disease or omphalitis is one of the economically important diseases of poultry. The affected chicks manifest depression, drooping of the head and huddling near to the heat source (Kahn et al., 2008). Several bacteria such as Escherichia coli, Salmonella species, Proteus species, Enterobacter species, Pseudomonas species, Klebsiella species, Staphylococcus species, Streptococcus species, Clostridium species, Bacillus cereus and Enterococcus have been isolated from the yolk sac of the infected birds in several studies (Corts et al., 2004; Iqbal et al., 2006). An outbreak of YSI caused by Klebsiella pneumonia in 1 to 3 day old canary chicks (Serinus canaria) have been reported recently (Razmyar and Zamani, 2016).
YSI is responsible for increased mortality in week-old chicks, poor weight gain, retarded growth and poor carcass quality in surviving birds (Corts et al., 2004). It may also result in decreased hatchability and increased culling rate due to retarded growth. It is accounting for large economic losses to the poultry industry with mortality rates reaching 5 to 10% (Ulmer, 2011). In Ethiopia, yolk sac infections (Omphalitis) have got little attention. Reports from Kombolcha hatchery in Ethiopia showed the significance of the problem with a prevalence rate of 33.1% (Abadi et al., 2013). Thus, the objectives of this study were to isolate and identify the bacteria from clinical cases of omphalitis (YSI) in newly hatched chicks from three selected hatcheries in Bishoftu Town and to determine their susceptibility against most commonly used antimicrobials.


Study area
The study was conducted on three selected hatcheries located in Bishoftu Town between November 2014 and June 2015. Bishoftu is located at 47 km Southeast of Addis Ababa at an altitude of about 1900 masl (38° 58′′ E 08° 44′′ N) harboring a number of commercial and small scale poultry farms. It has an annual rainfall of 1115.6 mm (NMSA, 2003). The three hatcheries were considered in this study due to the ease of access for sample collection and were designated as hatchery A, E and D for the purpose of this study.
Study animals and sampling
The chicks included in the current study were obtained from three successive batches of parent flocks of Lohmann (from hatcheries A and E) and Koekoek (Hatchery D) breeds. The chicks were physically examined for any signs of yolk sac infection with particular attention to the umbilical area and those with such signs were considered for further investigation. A total of 385 chicks (1 to 7 days old) showing signs of YSI were necropsied and examined for any gross lesions extending to the visceral organs with special reference to the yolk sac according to the procedures described previously (Chauhan and Roy, 2007). For bacteriological examination, yolk sac samples were collected aseptically using sterile plain swabs which were then labeled, packed and transported in portable coolant (ice pack) to Debre-zeit Agricultural Research Center Animal health laboratory. The collected samples were stored in refrigerator at 4°C until bacteriological analysis.
Bacterial isolation
Swab samples were aseptically streaked on to blood and MacConkey (Oxoid, UK) agar plates and incubated at 37°C for 16 to 40 h (Quinn et al., 2002) after which the plates were examined for any growth of bacteria. Based on macroscopic appearance, each of the different types of colonies observed were picked and sub-cultured to get pure cultures of each isolate. The isolates obtained were then streaked onto several selective agar plates including eosin methylene blue agar (EMB), brilliant green agar (BGA), mannitol salt agar (MSA), Salmonella-Shigella (SS) agar and Harlequin Salmonella ABC medium and incubated at 37°C for 16 to 40 h for further characterization.
Bacterial identification
Presumptive identification of bacterial isolates was done based on colony morphology (size, margin, elevation and color), Gram stain reaction and cellular morphology (Merchant and Packer, 1967) and biochemical characteristics using catalase, coagulase, M-R, V-P, Indole, triple sugar iron (TSI) agar and sugar fermentation tests as described previously (Swayne et al., 1998; Quinn et al., 2002; Cheesbrough, 2006).
Antimicrobial susceptibility test
Antimicrobials used for the current susceptibility test were selected based on their common application in the treatment of infections in poultry in Ethiopia which included Gentamicin, Amoxicillin, Penicillin, Kanamycin, Norfloxacin, Streptomycin, Chloramphenicol, Ciprofloxacin, Tetracycline, Sulfamethoxazole and Amikacin. Antimicrobial susceptibility was done by employing the disc diffusion or Kirby-Bauer method (Bauer et al., 1966). Briefly, an inoculum was prepared from an overnight culture of each bacterial isolate by suspending in sterile saline solution adjusted to 0.5 McFarland turbidity standard. Each isolate was inoculated onto previously prepared Mueller Hinton agar (Oxoid, UK) by streaking the whole surface of the plate with sterile cotton swab in a way to get evenly distributed confluent colonies. The inoculated MH plates were then made to dry and discs of selected antimicrobials were placed approximately 2.5 cm apart and gently pressed using sterile forceps. Plates were then incubated for 24 to 28 h at 37°C after which the diameter of zone of inhibition is measured and recorded. Determination of zone-size break points for defining the susceptible, intermediate and resistant categories for an antimicrobial agent was performed according to the established standards (NCCLS, 2007).
Data analysis
The laboratory result was coded and managed into Microsoft Excel and analyzed using statistical package for social sciences (SPSS) version 16. Descriptive analysis such as sum and percentages were used in summarizing the results. Chi-square test of independence was employed at 95% confidence level to determine whether significant differences exist in the rate of bacterial isolation among the three hatcheries.


Upon physical examination, all the birds included in the current study had the typical signs of omphalitis with a characteristic markedly thickened and dark blue navel, distended and soft abdomen. At necropsy, the major gross lesions observed in chicks with yolk sac infection were unabsorbed yolk sac; in many cases (370 out of 385) congestion and discoloration of the yolk (greenish yellow; dark brown to bright yellow), retained caseous yolk sac and edematous yolk (especially in 3 to 7 days old chicks) (Figure 1A and B). Peritonitis, pericarditis, petechial and ecchymotic hemorrhages on the serosal surface of visceral organs (particularly of the intestine) were also observed.

Bacterial isolation and identification from yolk sack specimens showed three predominant bacteria species which included Escherichia coli, Salmonella spp. and Staphylococcus aureus based on cultural, morphological and biochemical features which were consistent with the characteristics of the respective bacterial species (Table 1). The frequency of isolation of the predominant bacterial species from cases of omphalitis from the three hatcheries is as shown in Table 2. In all cases of yolk sac infections, more than one type of bacteria species was isolated revealing that mixed infection is a common scenario. Out of the total 323 different bacteria isolates belonging to the different genera obtained from cases of yolk sac infections from the three hatcheries, E. coli (N=116; 35.491%) was the most predominant isolate followed by Salmonella spp. (N=111; 34.36%) and S. aureus (N=96, 29.72).






Yolk sac infection is one of the health problems of poultry responsible for considerable losses. It results in decreased hatchability, increased mortality and culling rate in affected flocks due to retarded growth following alteration in structure of immunoglobulin proteins accompanied by microbial infection subsequently resulting in immunosuppression (Sander et al., 1998). It occurs mainly due to bacterial contamination of the egg shell after the egg is laid, while the cuticle is still moistened. Factors promoting contamination include lack of hygiene in the nests, presence of eggs on the floor, incubation of dirty eggs or eggs with egg shell defects and collection of dirty and clean eggs at the same time (Rahman et al., 2007; Ahmed, 2009; Ulmer, 2011).The current study carried out on the isolation of bacterial agents associated with yolk sac infection (omphalitis) in three hatcheries in Bishoftu is the first report in the study area which showed the importance of yolk sac infection as the cause of high mortality in chicks during their first week of life.
The findings of the three predominant bacterial species (E. coli, Salmonella and S. aureus) isolated from yolk sac infection in this study are corroborated by several earlier studies which documented the frequent association of these bacteria species with yolk sac infections (Amer et al., 2017; Abdel-Tawab et al., 2016; Hazariwala et al., 2002; Rosario et al., 2004; Iqbal et al., 2006; Buhr et al., 2006; Suha et al., 2008). E. coli has been previously reported as one of the most frequently isolated bacteria involved in the development of yolk sac infection (omphalitis) (Amer et al., 2017; Abdel-Tawab et al., 2016; Buhr et al., 2006; Suha et al., 2008). S. aureus has also been reported as an important cause of diseases in poultry (Hazariwala et al., 2002) as well as a common cause of yolk sac infection in broilers (Amer et al., 2017; McCullagh et al., 1998). Involvement of Staphylococcus, Proteus, Streptococcus and bacillus species has also been reported previously (Amer et al, 2017; Rosario et al., 2004; Buhr et al., 2006; Suha et al., 2008).
Several other species of bacteria have also been reported from cases of yolk sac infection in chicks including Salmonella, Staphylococcus, Protease, Bacillus, Streptococcus, Pseudomonas, Klebsiella, Clostridium, Aerobacter, Citrobacter, Achromobacter, and Enterococci spp. (Amer et al., 2017; Anjum, 1997; Deeming, 1995; Sainsbury, 1992). Involvement of Aspergillus fumigatus in yolk sac infection was also reported by Schonhofen and Garcia (1981).
The relatively few bacteria species isolated in the current study may be due to the use of antimicrobial agents by some of the poultry farms to control early chick mortality. Consistent to the current study, the gross lesions observed in chicks suffering from yolk sac infection which were manifested as edematous and unabsorbed/retained yolk sac was also reported by different workers (Suha et al., 2008; Ahmed et al., 2009; Kawalilak et al., 2010).
Several previous studies indicated that prolonged storage of eggs, improper temperatures and humidity during incubation as predisposing factors contributing to increased incidence of YSI. Too high or low incubation temperature during the final days of incubation will produce poorly closed navels. When eggs are stored for prolonged periods prior to incubation, more chicks with black scab navels are observed, ­indicating unhealed navels at the moment of hatching. Too high humidity during incubation results in insufficient weight loss. As a result, the residual yolk sac becomes enlarged, which prevents the navel from closing properly.
Conversely, when humidity is too low, the yolk sac ­dehydrates and becomes hard, which can damage sensitive tissue around the navel (Sainsbury, 1992; Sarma et al., 1985; Saif et al., 2003). Improper management associated with the conditions of egg storage and incubation may have played a role in predisposing young chicks for yolk sac infection observed in the current study requiring further investigation into the specific management practices in the hatcheries to have conclusive remark. The higher susceptibility of bacterial isolates obtained from YSI to antimicrobials such as Gentamicin, Chloramphenicol, Amikacin and Kanamycin in in-vitro drug sensitivity test were in agreement with the previous reports (Salehi and Bonab, 2006; Sharada et al., 2010) indicating their potential application for effective treatment of diseases caused by these bacteria species.
The resistance of E. coli, Salmonella and S. aureus to multiple antimicrobials observed in this study supports the reports of previous works (Khan et al., 2002; Nasrin et al., 2012; Lee et al., 2005; Abadi et al., 2013). This may probably suggest the wide use of these antimicrobials for treatment of bacterial infections in both animals and humans as these bacteria are environmental contaminants from clinical cases. This necessitates establishing alternative efficient strategies for the prevention and control of bacterial omphalitis through careful improvements of sanitary measures in hatcheries. In conclusion, the results of the present study in Bishoftu hatcheries from cases of omphalitis in chicks show that E. coli, Salmonella spp. and S. aureus, are the predominant bacteria isolated from yolk sack samples indicating that these bacteria species are the major cause of yolk sac infection.
Antimicrobials such as Gentamicin, Chloramphenicol, Kanamycin and Amikacin may be potentially effective for treatment of yolk sac infection in chicks. However, the isolation of multi-drug resistant strains of E. coli, S. aureus and Salmonella spp. from cases of chicks suffering from omphalitis is alarming as this resistance may spread to microbes infecting man and animals. The findings from the current study signify the importance of the problem requiring more detailed study to determine the extent of the problem in different poultry farms, the role and pathogenicity of each bacterial species involved in yolk sac infection as well as to explore possible predisposing factors.


The authors have not declared any conflict of interests.


Abdel-Tawab AA, Nasef SA, Ibrahim OA (2016). Bacteriological and molecular studies on bacteria causing omphalitis in chicks with regard to disinfectant resistance. Global Veterinaria 17 (6):539-545.


Amer MM, Elbayoumi KM, Amin Girh ZMS, Mekky HM, Rabie NS 2017). A study on bacterial contamination of dead in shell chicken embryos and culled one day chicks. Int. J. Pharm. Phytopharm. Res. 7(2):5-11.


Abadi A, Ali M, Ashenafi S, Shahid N, Haileleul N (2013). Yolk Sac Infection (Omphalitis) in Kombolcha Poultry Farm, Ethiopia. American-Eurasian J. Sci. Res. 8(1):10-14.


Ahmed MS, Sarker A, Rahman MM (2009). Prevalence of infectious diseases of broiler chickens in Gazipur district. Bangladesh J. Vet. Med. 7:326-331.


Anjum AD (1997). Poultry Diseases. Vet Ag Publications, Faisalabad-Pakistan, pp. 178-180.


Bauer AW, Kirby WMM, Sherri JC, Turck M (1966). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493- 496.


Buhr RJ, Northcutt JK, Richardson LJ, Cox NA, Fairchild BD (2006). Incidence of unabsorbed yolk sacs in broilers, broiler breeder roosters, white Leghorn hens, and Athens-Canadian random bred control broilers. Poult. Sci. 85(7):1294-7.


Chauhan HVS, Roy S (2007). Poultry Diseases Diagnosis and Treatment. New Age International Pvt Ltd Publishers; 3rd ed.


Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nded. London English Language Book Society. pp. 100-194.


Corts C, Isaies G, Cuello C, Floes J, Campos C (2004). Bacterial isolation rate from fertile eggs, hatching eggs, neonatal broilers with yolk sac infection. Rev. Lateno americana de Microbiolog. 46:12-16.


Deeming DC (1995). Possible effect of microbial infection on yolk utilization in ostrich chicks. J. Vet. Rec. 136:270-271.


Hazariwala A, Sanders Q, Hudson CR, Hofacre C, Thayer SG, Maurer JJ (2002). Distribution of staphylococcal enterotoxin genes among Staphylococcus aureus isolates from poultry and humans with invasive staphylococcal disease. Avian Diseases 46:132-136.


Iqbal M, Shah IA, Ali A, Khan MA, Jan S (2006). Prevalence and in vitro antibiogram of bacteria associated with omphalitis in chicks. Pakistan Vet. J. 26:94-96.


Razmyar J, Zamani AH (2016). An outbreak of yolk sac infection and dead-in-shell mortality in common canary (Serinus canaria) caused by Klebsiella pneumonia. Iran J Vet Res. 17(2):141-143.


Kahn CM, Line S, Aiello SE (2008). In Merck Veterinary Manual. 9thed. Merck and Co, Inc., USA, pp. 2258-2259.


Kawalilak LT, Ulmer-Franco AM, Fasenko GM (2010). Impaired intestinal villi growth in broiler chicks with unhealed navels. J. Poult. Sci., 89:82-87.


Khan KA, Khan SA, Hamid S, Aslam A, Rabbani M (2002). A study on the pathogenesis of yolk retention in broiler chicks. Pakistan Vet. J. 22:175-180.


Lee YJ, Kim AR, Jung SC, Song SW, Kim JH (2005). Antibiotic resistance pattern of E. coli and Salmonella spp. isolated from chicken feces. Korean J. Vet. Res. 45:75-83.


McCullagh JJ, McNamee PT, Smyth JA, Ball HJ (1998). The use of pulsed field gel electrophoresis to investigate the epidemiology of Staphylococcus aureus infection in commercial broiler flocks. J. Vet. Microbiol. 63:275-281.


Merchant IA, Packer RA (1967). Veterinary bacteriology and virology. 7thed. The Iowa State University Press, Ames, Iowa, USA. pp. 211-305.


Nasrin S, Islam MA, Khatun ML, Akhter S (2012). Characterization of bacteria associated with omphalitis in chicks. Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Bangladesh Vet. J. 29(2):63-68.


National Committee for Clinical Laboratory Standards (NCCLS) (2007). Performance standards for antimicrobial susceptibility testing; 17th informational supplement. 27 M100- S17, NCCLS, Wayne, PA.


National Meteorological Service Agency (NMSA) (2003). Rainfall and temperature data, Addis Ababa, Ethiopia.


Quinn PJ, Markey BK, Carte ME, Donnelly WJ, Leonard FC (2002). Veterinary Microbiology and Microbial Disease. 1st ed. Cornwall, Great Britain. Blackwell Science Ltd., pp. 43-122.


Rosario C, Téllez I, López C, Villaseca F, Anderson R, Eslava C (2004). Bacterial isolation rate from fertile eggs, hatching eggs and neonatal broilers with yolk sac infection. Revista Latino Americana de Microbiología, 46:12-16.


Rahman MM, Rahman AZ, Islam MS (2007). Bacterial diseases of poultry prevailing in Bangla. J. Poultry Science 1:1-6.


Saif YM, Barnes HJ, Fadly AM, Glisson JR, Mcdougald LR, Swayne DE (2003). Diseases of poultry. 11th Edition CD-Rom version. Iowa State press, USA.


Salehi TZ, Bonab SF (2006). Antibiotics susceptibility pattern of Escherichia coli strains isolated from chickens with coli-septicemia in Tabriz Province, Iran. Int. J. Poult Science. 5(7):677-684.


Sainsbury D (1992). Poultry Health and Management. 3rd ed., Blackwell Scientific Publications, London, U.K. P 116.


Sander JE, Willinghan EM, Wilson JL, Thayer SG (1998). The effect of inoculating Enterococcus faecalis into the yolk sac on chick quality and maternal antibody absorption. Avian Dis. 42:359-363.


Sarma DRL, Char NL, Rao MRK, Khan DJ, Narayan G (1985). A comprehensive study on bacterial flora isolated from yolk sac infection in chicks. Indian J. Poult. Sci. 20:262-266.


Sharada R, Ruban WS, Thiyageeswaran M (2010). Isolation, characterization and antibiotic resistance pattern of Escherichia coli isolated from poultry. American-Eurasian J. Sci. Res. 5(1):18-22.


Schonhofen, CA, Garcia, RGF (1981). Aspergillus omphalitis in chicks. Arquivos de biologia-e-Tecnologia, 24(4):437-438.


Suha AH, Ali HH, Rizgar RS (2008). Bacteriological and pathological study of yolk sac infection in broiler chicks in Sulaimani district. Kurdistan 1st Conference on Biological Science. University of Dohuk 2-4, May, 2006. J. Dohuk. Univ.11:48-55.


Swayne DE, Glisson JR, Jack wood MW, Pearson JE, Reed WM (1998). A laboratory manual for the isolation and identification of avian pathogens. Pennsylvania, USA. American. Association of Avian Pathologists. University of Pennsylvania. 4th Ed. pp. 4-16.


Ulmer FAM (2011). Yolk Sac Infections in Broiler Chicks: Studies on Escherichia coli, Chick Acquired Immunity and Barn Microbiology. PhD thesis, University of Alberta Edmonton, Alberta. pp. 1-197.