Embryo mortality and Isolation of Escherichia coli as cause of death for in-shell chick embryos and first week chicks

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1 Embryo mortality and Isolation of Escherichia coli as cause of death for in-shell chick embryos and first week chicks Hailu Mazengia 1, Sefinew Alemu 2*, G. Mekuriaw 1 and Zewdu Wuletaw 1 1 College of Agriculture and Environmental Sciences, Bahir Dar University, Bahir Dar, Ethiopia 2 Department of Clinical Medicine and Epidemiology, Faculty of Veterinary Medicine, University of Gondar, P.O.Box 196, Gondar, Ethiopia *Corresponding author Abstract A study on embryo mortality and isolation E. coli from dead-in-shell chick embryos and first week chicks was carried out at Andassa Poultry Farm, Ethiopia. Hatchery data analysis, bacterial isolation, and pathogenesity tests were employed. In the retrospective study, chick embryo mortality rates of 23.63±1.69%, 26.95±1.16%, and 30.72±1.66% were observed in years 2006, 2007and 2008, respectively; and was statistically significant difference (p< 0.05) among the years with overall embryo mortality rate of 27.48±0.89%. Embryo mortality rate of 30.18±1.40%, 29.38±2.14%, 23.11±1.45% and 25.47±2.55% was observed in dry, early rainy, rainy and late rainy seasons, respectively with statistically significant difference (p<0.05) in rate among seasons. Embryo mortality rate in Rhode Island Red breeds (32.89±1.29%) was significantly higher (p< 0.05) than that of the Lohmann White breeds (22.86±0.97%). Interaction effect of year with season and year with breed were both significant (p<0.05) on embryo mortality rate. Year, season and breed observed significant interaction effect on embryo mortality rate. Escherichia coli were isolated from 85.71% of dead-in-shell chick embryos and 54.17% of first week chicks, gross lesions. Escherichia coli were isolated at highest frequency from dead-in-shell embryos in early rainy 18/20 (90.00%) followed by dry 37(89.19%), rainy 7(44.67%), and late rainy 14 (73.68%), seasons with significant difference (p<0.05) in frequency of isolation among seasons. The study indicated need of hygienic egg selection and hygienic hatchery management. Comprehensive study to identify factors contributing for death of in-shell chick embryos and first week chicks is suggested. Key words: Andassa; Chick; Embryo; Escherichia coli; In-shell, Mortality 1

2 INTRODUCTION Avian collibacillosis, caused by Escherichia coli, has been noticed to be a major infectious disease in birds of all ages and has an important economic impact on poultry production worldwide. The majority of economic losses result from mortality and decrease productivity of the affected birds (Oaky, 1995). Fecal contamination of eggs may result in the penetration of E. coli through the shell and may spread to the chicks during hatching and is often associated with high mortality rates, or it may give rise to yolk sac infection. In association with various disease conditions, E. coli results in heavy economic losses either as primary or as a secondary pathogen (Calnek et al., 1997). In poultry, it has a variety of disease manifestations including yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis and salpingitis. In its acute forms, it is characterized by septicemia resulting in death and by peri-carditis, airsacculitis and peri-hepatitis in its subacute forms (Calnek et al., 1997). As poultry play an important role in the diet and economy of Ethiopia (Solomon, 2004), emphasis has been given to the sector and use of improved exotic breeds is increasing in recent times (Alemu and Tadele, 1997). In this regard, bureau of Agriculture and Rural development of Amhara Regional State launched and implemented poultry development strategy to raise income by distributing day-old-chicks to selected farmers. Day-old-chicks of Rhode Island Red (RIR) and Lohmann white (LOH) are being distributed from two separate hatcheries with capacity of 6300 and 1830, respectively. However, in-shell embryonic mortality and mortality in first week chicks is important threat for this practice for which infectious problems including collibacillosis are of major problems in many of poultry farms in the country. Avian colibacillosis, caused by Escherichia coli, is one of the principal causes of morbidity and mortality. Chanie et al. (2009) isolated Escherichia coli from the blood, liver and joint samples in an outbreak of a disease in broiler chicks in one of poultry farms in Central Ethiopia. Therefore, the present study was aimed to identify the causes of dead-in-shell chick embryos and dead in first week chicks in different hatches within hatcheries at Andassa Government poultry farm and to suggest appropriate control and prevention strategies for the particular farm. MATERIAS AND METHODS 2

3 Study area This study was conducted in Andassa poultry farm, an area located 605 kilometer northwest of the capital Addis Ababa. The elevation of the area is 1780 masl. The annual temperature and rainfall in the study area varies from 9-34 C and mm, respectively. The study period was classified in to four seasons. The dry and early seasons last from November to April and from May to June whereas the rainy and late rainy seasons extend from July to August and from September to October, respectively (CSA, 2006). Study animals and their management During the study period, parent flocks of two breeds, Lohmann white and Rhode Island Red. The parent flocks were kept in separate rearing houses with an average distance of 15 to 30 meters between them. The flocks have their own attendants who provide feed and water, clean the equipments and collect eggs daily from laying houses to cooling rooms. They were vaccinated against Salmonella pollurum and Salmonella gallinarum using polyvalent vaccine from the National Veterinary Institute of Ethiopia. Parent flocks were provided with formulated ration from government owned feed processing industry, Kality, twice daily, and water adlib. Eggs were collected twice a day before noon at 4:00 local time and early in the afternoon at 8:00, immediately displayed in the cooling room at least for 10 days before setting into the incubator. The eggs used for hatching were disinfected by fumigation with formalin and potassium permanganate (1gm KMnO 4 /2 ml formalin) before being stored at 15 C. Second fumigation was made at the day 18 th of hatch. River water after it has been treated with sodium hypochloride is used to wash the incubator and the setter during incubation. Study design and sampling method Retrospective hatchery data analysis and culturing of E. coli were conducted from dead-in-shell chick embryos and dead in first week chicks. A total of 139 samples, 91 from dead-in-shell and 48 from dead first week chicks, were collected from the hatchery of Andassa Government Poultry Farm in different seasons. In addition, three years retrospective data which was obtained from the hatchery records was used to study quantify dead-in-shell embryos. 3

4 Bacterial isolation Specimens were directly inoculated on Blood and MacConkey Agar media and incubated aerobically at 37 C for 24 hr at Bahir Dar Regional Veterinary Laboratory; the colonies were examined with the naked eye for their cultural characteristics, morphological properties and any changes in the media. Then the organism was stained with Gram ' s Method. Identification of the isolates was performed according to methods described by Swayne et al. (1998) and Margie and Lawrence (1999). Pathogenecity test For conducting pathogenesity test of the isolated Escherichia coli, the isolated colonies were propagated for three days to increase the number of colonies. Then the colonies were counted and dissolved with buffered saline solution of 0.5 cc at a dose of The suspension was administered for eighteen 4- weeks old (9 for each) pullets through caudal air sac route according to Chauhan and Roy, (1998) and Fowler (2001). The inoculated chicks were followed for 72 hours for presence of clinical signs. Data analysis Hatchery data on embryo mortality rate was analyzed using least squares procedures of the General Linear Model of analysis system using Statistical Procedure for Social Sciences (SPSS Version 16, 2009). The fixed effects considered in this study were the following parameters: breed (Rhode Island Red, Lohmann White), Season (dry, prerainy, rainy and post rainy) and year of hatch (2006, 2007 and 2008). When analysis of variance declares significance, least square means were separated using adjusted Tukey-Kramer test. The model for Analysis of variance of embryo mortality rate was: E ijk = µ + Y i + S j + B k + e ijk Where: E ijklm = the observation on embryo mortality. µ = over all mean B i = Fixed effect of year (i = 2006, 2007 and 2008) S j = Fixed effect of season (j = dry, prerainy, rainy and post rainy) B k = Fixed effect of breed (k = Rhode Island Red and Lohmann White) Y i S j = the interaction effect of year and season 4

5 Y i B k = the interaction effect of year and breed Y i S j B k = the interaction of year, season and breed e ijk = effect of random error RESULTS Analysis hatchery data during the study period indicated overall embryo mortality rate of 27.48±0.89%. Significant difference (p< 0.05) in embryo mortality rates among years was observed. The highest embryo mortality was observed in the early rainy season while the lowest was observed in the late rainy season, and there was significant difference (p< 0.05) in embryo mortality rates among seasons. The embryo mortality rate in Rhode Island Red breeds was significantly higher (p< 0.05) than that of the Lohmann White chick (Table 1). The interaction effect of year with season was found significant (p<0.05) on embryo mortality rate. In the same scenario, the interaction effect of year with breed was found significant (p<0.05) on embryo mortality rate. The interaction effect of year, season and breed was also found significant effect on embryo mortality rate. Gross pathological examination of dead-in-shell and dead first week chicks indicted lesions that are suggestive of Escherichia coli which include airsaculitis, unabsorbed yolk and pericarditis. Escherichia coli were isolated from gross lesion samples collected from dead-in-shell chick embryos and first week chicks. Escherichia coli were isolated from 78 of 91 (85.71%) of dead-in-shell embryos, and isolated at highest frequency in the early rainy season followed by the dry season. Frequency of Escherichia coli isolated from dead-in-shell chick embryos among seasons was statistically significant difference (p<0.05). On the other hand, from the total 48 dead In first week chicks sampled, Escherichia coli were isolated from 26 of 48 (54.17%) dead in first week chicks, and there was no statistically significant difference (p>0.05) in frequency of Escherichia coli isolated from dead in first week chicks seasons (Table 2). The pathogenesity test for isolated Escherichia coli after inoculation to pullets showed signs like roughened feather, mild diarrhea and inappetance. Necropsies on dead chicks were made after 48 hours to view the gross lesions. But no any prominent lesions were observed except congestion of air sac and 5

6 pericardium. Sample collected from air sac and pericardial sac were collected and cultured and Escherichia coli colonies were isolated. DISCUSSION The overall embryo mortality rate from analysis hatchery data during the study period was 27.23±0.76%, which was in agreement with the work of Bungo et al. (2011) who reported 26.7% embryo mortality in Hinai-dori native Japanese chick breeds. However by the same study, Bungo and his colleagues in other native Japanese chick breeds. There were significant differences both in the magnitude and frequency of embryo mortality rates among the study seasons. The increased in magnitude and frequency of embryo mortality rate during dry and early rainy than rainy and late rainy seasons may be associated with the higher level of contamination of the river water during these seasons (Getinet, 2003). Bacterial cultures from both deadin-shell chick embryos and dead in first week chicks revealed Escherichia coli infection. This finding is in agreement with the work of Cortes et al. (2004) who reported embryo mortality rate is mostly associated with yolk sac infections and omphalitis due to presence of Escherichia coli in water which can contaminate egg shells. The isolation rate of Escherichia coli from dead-in-shell embryos in the early rainy, dry, rainy and late rainy seasons by the current study was in agreement with the work of Kabilika and Sharma (1997), and Raji et al. (2003), who isolated Escherichia coli predominantly from dead-in-shell embryos, although percentage of Escherichia coli isolates varied between different authors. The overall frequency of Escherichia coli isolated in first week chicks was lower than the work of Cortes et al. (2004) who isolated Escherichia coli at frequency of 45.50% from broilers in Mexico. This might be due to difference in the method of isolation or due to difference in prevalence of Escherichia coli in different areas or due to difference in management practices applied in the respective study areas. Use of river water for the poultry farm activities can be sited as best example in this regard. According to Chen et al. (2002) and Singh (2002) management practices associated with storage, disinfection, and setting of eggs in the hatchery are important predisposing factors for Escherichia coli infection of chicks if they are below the standard. In summary, the present study indicated that first 6

7 week chicks and died-in shell embryos were found harboring Escherichia coli, which can be the root cause for death. Indirect effect of Escherichia coli on growth and feed utilization might also be causing significant economic loss, but left unnoticed. Therefore, hygienic practice especially during hatchery management and egg selection should be applied. In addition, the management of the farm should give emphasis to use clean water and to reduce water flooding in the area. Finally, further study is suggested to identify other factors contributing for the death of in-shell chick embryos and first week chicks in the farm. Acknowledgment The authors would like to acknowledge the staff of Andassa poultry farm especially those working in the hatchery. Administration of Bahir Dar Regional Veterinary Laboratory deserves thanks for their cooperation and allowing use of laboratory facilities. REFERENCES Alemu, Y. and D. Tadelle, Status of poultry research and development in Ethiopia. Research Bulletin No.4. Debre Zeit Agricultural research centre, Alemaya University of Agriculture, pp. 63. Bungo, T., T. Goto, J. Shiraishi and M. Tsudzuki, Embryonic and chick mortality of four native Japanese Chick Breeds. Journal of Animal and Veterinary Advances, 10: Calnek B., H. Barnes, C. Beard, L. McDougal and Y. Saif, Diseases of Poultry. 10th ed. Iowa State University Press; Ames, IA, USA. Central Statistical Agency (CSA), Report on livestock resources of Amhara Region, Pp Chanie, M., T. Negash, and B. S. Tilahun, Occurrence of concurrent infectious diseases in broiler chicks is a threat to commercial poultry farms in Central Ethiopia, Trop Anim Health Prod, 41: Chauhan and Roy, Poultry diseases& prevention methods, New Age International Limited Publishers. India Pp Chen, S. J., M. E. Wang, J. T. Cho, and H. C. Wang, Monitoring the hygiene of chick hatcheries in Taiwan during Journal of Microbiology and Immunology, 35: Cortes, C., G. Isaias, C. Cuello, J. Flores and R. Anderson, Bacterial isolation rate from fertile eggs, hatching eggs and neonatal broilers with yolk sac infection. Journal of Avian Diseases, 43:

8 Fowler, N. G., How to carry out field investigation, in Poultry Diseases, 4th edn., F. Jordan, T. Pattison, W. & M., Eds. Saunders, London, Pp Getinet, Z., Artificial insemination in poultry, MSc Thesis, Alemaya`University of agriculture. Kabilika, H. and R. Sharma, Escherichia coli from Dead in shell embryos from hatcheries in Zambia. Bulletin of Animal. Health Production Africa, 45, Margie, D., and H. Lawrence, A laboratory manual for the isolation and identification of avian pathogens. 4th ed. American Association of Avian Pathologists, Athens, GA. Oaky, Y., Poultry disease control program in Japan. Asian livestock Research for rural Development 16(2): Raji M., J. Adekeye, J. Kwaga and J. Bale, In vitro and in vivo pathogenicity studies of Escherichia coli isolated from poultry in Nigeria, Israel Journal of Veterinary Medicine, 58 (1):1-5 Singh, R., Poultry production. Kalyani Publishers, Newdelh-Ludhiana, Pp.111. Solomon, D., Egg production performance of local and White Leghorn hens under intensive and rural household conditions of Ethiopia. Jimma college of Agriculture, Pp Statistical Package for Social Science (SPSS), 2009, Release The Apasche software foundation. Swayne, D., J. Gilson, M. Jack wood, J. Pearson, and W. Reed, Laboratory manual for isolation and identification of avian pathogens. 4 th ed., American Association of Avian Pathologist, inc., Kennett square, Pennsylvania, USA. 4. 8

9 Table1: Least square means and standard errors on embryo mortality rates Source of variation No. of embryos LSM±SE (%) Year ±1.74 a 26.78±1.13 b 30.73±1.16 b Season Dry ±1.77 c Early rainy ±1.05 c Rainy ±1.84 d Late rainy ±1.34 d Breed RIR ±1.02 e LOH ± 1.1 f Total ±0.76 Numbers in each column with different letters indicate significant difference Table 2: Number of samples and frequency of Escherichia coli isolated from dead-in-shell chick embryos and from first week chicks Season Dead-in-shell chick embryos First week chicks No. examined Positive (%) 95%CI No. examined Positive (%) 95%CI Dry (89.19) (70.58) Early rainy (90.00) (53.84) Rainy 15 7 (46.67) (44.44) Late rainy (73.68) (33.33) Total (79.12) (54.16)