1. broiler chickens بسم الله الرحمن الرحيم

2.  Pathogenesis of Enterobacteriaceae isolated from commercial chicken eggs in broilers *M.M.Amer, Sherein, S.abdelgayed, A. Hussien, M.Dahshan, and Asmaa A.Mohamed

3. Introduction  Enterobacteriaceae is a family of rod-shaped, aerobic, and facultative anaerobic bacteria.  The Enterobacteriaceae family is subdivided into 8 tribes including: Escherichieae, Edwardsielleae, Salmonelleae, Citrobactereae, Klebsielleae, Proteeae, Yersineae, and Erwineae.

4. Introduction  Fertilized eggs contaminated with micro- organisms may result in weak chicks, poor chick growth and low feed conversion rate.  Proteus sp., Enterobacter sp., Pseudomonas sp., Klebsiella sp., Staphylococcus sp., Streptococcus sp., Clostridium sp., Bacillus cereus, S. typhimurium and Enterococcus has been isolated from hatching eggs. However, the most common isolated bacterium is E. coli.

5. Purpose of the study  This study was done to investigate the pathogenicity of some Enterobacteriaceae isolates isolated from chickens and eggs ;  Through different Performance parameters including;,,,.  Clinical signs and /or mortality, postmortem lesions, food consumption and body weight, as well as histopathological examination.

6. Materials and Methods  I- Experimental Chicks:  One hundred and fifty 1 day old broiler hybrid chicks were purchased from Cairo Co. and raised on non medicated diet free from anticoccidial and antibiotic on deep litter. Feed and water were consumed ad-libitum with continous source of light.

7. Materials and Methods  II-Bacterial-isolates :  E. coli, E. Sakazakii, E. fergusonii, and P. mirabilis previously isolated according to Amer et.al. (2013).  Subcutaneously inoculations of 0.5 ml suspension of previously mentioned isolates using McFarland standard.  Then the birds were kept under daily observation for clinical signs, mortality, and post mortem lesions

8. Materials and Methods III- Experiment design  III- Experiment design  150 one day old broiler hybrid chicks were divided into 5 groups. GroupsNumberInfected groups

9. Materials and Methods IV- Chicken performance IV- Chicken performance Daily observation of all groups including;  The clinical signs and /or mortality,  Postmortem lesions,  Food consumption, and  Body weight were recorded.

10. Materials and Methods V- Histopathological examination 3 rd 6 th 9 th 12 th 15 th 3  Tissue samples were taken from each group at 3 rd,6 th, 9 th, 12 th, and 15 th day post infection (dpi) from liver and intestine of 3 sacrificed birds from infected and control chicken groups immediately after cervical dislocation.  All the specimens were fixed, processed, and routinely stained with hematoxyline and eosin for histopathological examination.

11. Results and Discussion  This experiment was carried out to investigate the pathogenicity of Enterobacteriacea isolates from commercial chicken eggs; both table and fertilized type. E coliE Sakazakii, E. fergusonii P. mirabilis  Enterobacteriaceae used in this study includes E coli, E Sakazakii, E. fergusonii and P. mirabilis.

12. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 1  Group 1 (infected with E.coli isolate):  The main clinical symptoms were respiratory signs (40%), and pasty vent (30%).  The mortality rate during the experiment was 27.5% at different intervals.

13. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 1  Group 1 (infected with E.coli isolate): Group No infection Age / week Mean Weekly body weight /gm Mean Weekly feed consumption /gm MeanWeekly body weight gain /gm FCR 1E.coli 1 90±48 1261380.9 2 201 ± 69 382.21512.5 3 286 ± 103 3642231.6 5 Control negative 1 90±48 1261380.9 2 305±77 417.22101.9 3616±1256204401.4

14. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 2  Group 2 (infected with E.Sakazakii isolate):  The clinical signs were;  dullness, depression, sleepy and ruffled feather.  These clinical signs gradually developed to become brown diarrhea, enlarged shank, and coughing from 2 nd to 4 th dpi,  But at 5 th dpi one chick died.

15. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 2  Group 2 (infected with E.Sakazakii isolate): Group No infection Age / week Mean Weekly body weight /gm Mean Weekly feed consumption /gm MeanWeekly body weight gain /gm FCR 2E. Sakazakii 1 90 ± 48 1261380.9 2 298 ± 70 382.21562.5 3 468 ± 88 4532651.7 5 Control negative 1 90±48 1261380.9 2 305±77 417.22101.9 3616±1256204401.4

16. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 3  Group 3 (infected with E. fergusonii isolate) :  At the 1 st dpi, clinical signs described as dullness, depression, white and greenish diarrhea in 5 (17%) chicks,  These clinical signs gradually developed to respiratory signs as coughing, sneezing and ralls from the 2 nd to 7 th dpi.

17. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 3  Group 3 (infected with E. fergusonii) isolate): Group No infection Age / week Mean Weekly body weight /gm Mean Weekly feed consumption /gm MeanWeekly body weight gain /gm FCR 3E. fergusonii 1 90 ± 48 1261380.9 2 303 ± 74 307.5171.51.8 3 457 ± 56 262.51292 5 Control negative 1 90±48 1261380.9 2 305±77 417.22101.9 3616±1256204401.4

18. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 4  Group 4 (infected with P. mirabilis isolate):  1 st day post infection, the clinical signs described as dullness, depression, and brown to orange diarrhea in 4 (13%) chicks,  These clinical signs gradually developed to respiratory signs as coughing, sneezing, Ralls and also brown diarrhea from the 2 nd to 7 th dpi.  The mortality rate during the experiment was 55% at different intervals.

19. Results and Discussion I-Chicken performance results  I-Chicken performance results  Group 4  Group 4 (infected with P. mirabilis isolate): :: Group No infection Age / week Mean Weekly body weight /gm Mean Weekly feed consumption /gm MeanWeekly body weight gain /gm FCR 4P. mirabilis 190±481261380.9 2294 ± 68237.71281.8 3503 ± 95480.53061.6 5 Control negative 1 90±48 1261380.9 2 305±77 417.22101.9 3616±1256204401.4

20. Results and Discussion II- II- Pathological results Group 1 Group 1 (infected with E.coli isolate): Postmortem lesions Postmortem lesions:  Congestion of liver, spleen, kidneys and lungs,  pericarditis, perihepatitis and air sacculitis.

21. Results and Discussion II- Pathological results Group 1 (infected with E.coli isolate): Histopathological findings :  Histopathological findings : (1) Liver (1) Liver

22. Group 1 (infected with E.coli isolate): Group 1 (infected with E.coli isolate): Focal areas of hepatic necrosis infiltrated by mononuclear cells (H&EX 400) At 6 th and 9 th dpi

23. Group 1 (infected with E.coli isolate): Group 1 (infected with E.coli isolate): Portal leucocytic cell infiltration (H&E X 400). 15 th dpi,

24. Results and Discussion II- Pathological results Group 1 (infected with E.coli isolate): Histopathological findings :  Histopathological findings : (2) Intestine (2) Intestine

25. Group 1 (infected with E.coli isolate): Group 1 (infected with E.coli isolate): At 3 rd dpi Slight activation of mucous secreting glands (H&E X 200).

26. Group 1 (infected with E.coli isolate): Group 1 (infected with E.coli isolate): At 6 th and 9 th dpi Sub mucosal leucocytic infiltration(H&E X 400).

27. Results and Discussion II- II- Pathological results Group 2 Group 2 (infected with E.Sakazakii Postmortem lesions Postmortem lesions:  Septicemic picture from the 1 st to the 4 th dpi represented by congested lung and Spleen, air sacculitis, hepatitis with streaks of hemorrhages on its surface,  distended gall bladder, congested kidney, slight to moderate pericarditis, petechial hemorrhage on coronary fat, endocardium and on brisket muscle.

28. Results and Discussion II- Pathological results II- Pathological results Group 2 Group 2 (infected with E.Sakazakii Histopathological findings :  Histopathological findings : (1) Liver (1) Liver

29. Group 2 Group 2 (infected with E.Sakazakii Large focal area of hepatic necrosis infiltrated with leucocytic cells (H&E X 400). At 3 rd and 6 th dpi

30. Group 2 Group 2 (infected with E.Sakazakii Portal tract permeation with leucocytic cell infiltrations (H&E X 400). At 9 th dpi

31. Results and Discussion II- Pathological results Group 2 Group 2 (infected with E.Sakazakii Histopathological findings :  Histopathological findings : (2) Intestine (2) Intestine

32. Group 2 Group 2 (infected with E.Sakazakii Massively degenerated mucosa (H&E X 200). At 3 rd and 6 th dpi.

33. Group 2 Group 2 (infected with E.Sakazakii Massive sub mucosal gland necrosis (H&E X 400).. At 9 th dpi

34. Results and Discussion II-  II- Pathological results Group 3 Group 3 (infected with E. Fergusonii isolates); Postmortem lesions Postmortem lesions:  Septicemic picture from the 1 st to 4 th dpi appeared as congested lung, air sacculitis and spleen, slight to moderate pericarditis, and enlarged distended ceci with gases.  Multiple serosal petechial hemorrhages and fibrinous peritonitis were present.

35. Results and Discussion II- Pathological results Group 3 Group 3 (infected with E. Fergusonii isolates); Histopathological findings :  Histopathological findings : (1) Liver (1) Liver

36. Group 3 Group 3 ( infected with E. Fergusonii isolates ); Portal tract dilated and congested vessels (H&E X 400). At 9 th and 12 th dpi.

37. Group 3 Group 3 ( infected with E. Fergusonii isolates ); Focal area of necrosis with leucocytic infiltration (H&E X 400). At 15 th dpi.

38. Results and Discussion II- Pathological results Group 3 Group 3 (infected with E. Fergusonii isolates); Histopathological findings :  Histopathological findings : (2) Intestine (2) Intestine

39. Group 3 Group 3 ( infected with E. Fergusonii isolates ); Sub mucosal mononuclear cells infiltration (H&E X 400). 3 rd and 6 th dpi

40. Group 3 Group 3 ( infected with E. Fergusonii isolates ); Hyperactivity of the glands (H&E X 200).. 9 th, 12 th, and 15 th dpi

41. Results and Discussion II-  II- Pathological results Group 4 Group 4 (infected with Pr.mirabilis isolates) ; Postmortem lesions Postmortem lesions:  Severe septicemic picture from the 1 st to 4 th dpi appeared as congested lung and spleen,  air sacculitis, distended gall bladder, congested kidney, severe pericarditis, diffuse petechial hemorrhage on coronary fat, endocardium and on brisket muscle,

42. Results and Discussion II- Pathological results Group 4 Group 4 (infected with Pr.mirabilis isolates) ; Histopathological findings :  Histopathological findings : (1) Liver (1) Liver

43. Group 4 Group 4 ( infected with Pr.mirabilis isolates ) At 3 rd, 6 th, and 9 th dpi Focal areas of hepatic necrosis infiltrated with leucocytic cells (H&E X 400).

44. Group 4 Group 4 ( infected with Pr.mirabilis isolates ) Hyperplasia of epithelial lining bile duct, cholangitis, newly formed bile ductules, and massive portal leucocytic cell ifiltration (H&E X 200). At 12 th and 15 th dpi

45. Results and Discussion II- Pathological results Group 4 Group 4 (infected with Pr.mirabilis isolates) ; Histopathological findings :  Histopathological findings : (2) Intestine (2) Intestine

46. Group 4 Group 4 ( infected with Pr.mirabilis isolates ) Severly necrotic glands (H&E X 200). At 3 rd, 6 th, and 9 th dpi

47. Group 4 Group 4 ( infected with Pr.mirabilis isolates ) Submucosal leucocytic infiltration (H&E X 400).. At 12 th and 15 th dpi

48. Conclusion  It could be deduced that;  the isolates of Enterobacteiacea were highly pathogenic.  From the histopathological point of view, it is considered that Pr.mirabilis is the most pathogenic followed by E. Sakazakii, E. Fergusonii, and E. coli respectively.  Moreover, it is recommended to take these bacterial types in consideration in taking preventive and control measures from the poultry and public health points of view.