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Prevalence	
  and	
  molecular	
  characterisa2on	
  of	
  
Eimeria	
  species	
  in	
  Ethiopian	
  village	
  chickens	
  
L.	
  Luu1	
  ,	
  J.	
  Be*ridge1,2,	
  R.	
  Christley1,	
  K.	
  Melese3,	
  	
  D.	
  Blake4,	
  T.	
  Dessie2,	
  P.	
  Wigley1	
  ,	
  T.	
  Desta2,5	
  ,	
  O.	
  Hano*e,5,	
  P.	
  Kaiser6,	
  Z.	
  Terfa2,7,	
  	
  
M.	
  Collins1	
  and	
  S.	
  Lynch1,2	
  	
  
	
  
1	
  Ins&tute	
  of	
  Infec&on	
  and	
  Global	
  Health,	
  University	
  of	
  Liverpool,	
  UK;	
  2	
  Interna&onal	
  Livestock	
  Research	
  Ins&tute,	
  Addis,	
  Ababa,	
  Ethiopia;	
  3	
  Debre	
  Zeit	
  
Agricultural	
  Research	
  Centre,	
  Ethiopian	
  Ins&tute	
  for	
  Agriculture	
  Research,	
  Debre	
  Zeit,	
  Ethiopia;	
  4	
  Department	
  of	
  Pathology	
  and	
  Infec&ous	
  Diseases,	
  
Royal	
  Veterinary	
  College,	
  UK;	
  5	
  Centre	
  for	
  Gene&cs	
  and	
  Genomics,	
  School	
  of	
  Biology,	
  University	
  of	
  NoPngham,	
  UK;	
  6	
  The	
  Roslin	
  Ins&tute	
  and	
  Royal	
  
(Dick)	
  School	
  of	
  Veterinary	
  Science,	
  University	
  of	
  Edinburgh,	
  UK;	
  7	
  Management	
  School,	
  University	
  of	
  Liverpool,	
  UK	
  
	
  
	
  
	
  
	
  
Sampling	
  Strategy	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
Eimeria	
  
	
  
•  Protozoan	
  parasites	
  are	
  found	
  worldwide	
  and	
  	
  in	
  all	
  types	
  of	
  
chicken	
  producOon	
  system	
  
•  7	
  species	
  are	
  known,	
  but	
  are	
  difficult	
  to	
  differenOate	
  based	
  	
  on	
  
appearance	
  under	
  the	
  microscope	
  
•  Some	
  species	
  cause	
  severe	
  disease	
  and	
  death,	
  whilst	
  others	
  	
  are	
  
only	
  mildly	
  pathogenic,	
  but	
  may	
  reduce	
  growth	
  rates	
  
References	
  
1.	
  Permin	
  A,	
  Hansen	
  JW:	
  Epidemiology,	
  diagnosis	
  and	
  disease	
  control	
  of	
  poultry	
  parasites.	
  Food	
  and	
  Agriculture	
  OrganizaOon	
  of	
  the	
  United	
  NaOons:	
  Rome;	
  1998.	
  
2.	
  Vrba	
  V,	
  Blake	
  DP,	
  Poplstein	
  M:	
  Quan2ta2ve	
  real-­‐2me	
  PCR	
  assays	
  for	
  detec2on	
  and	
  quan2fica2on	
  of	
  all	
  seven	
  Eimeria	
  species	
  that	
  infect	
  the	
  chicken.	
  Vet	
  Parasitol	
  2010,	
  174:183–190.	
  
3.	
  McDougald	
  LR:	
  Coccidiosis.	
  In	
  Diseases	
  of	
  poultry.	
  11th	
  ediOon.	
  Edited	
  by	
  Saif	
  YM,	
  Barnes	
  J,	
  Glisson	
  JR,	
  Fadly	
  AM,	
  McDougald	
  LR,	
  Swayne	
  DE.	
  Ames:	
  Iowa	
  State	
  Press	
  ;	
  2003.	
  
	
  
Roundworm	
  
egg	
  
Eimeria	
  oocyst	
  
Eimeria	
  oocysts	
  
X100	
  magnifica2on	
   X200	
  magnifica2on	
  
Methods	
  
	
  
•  Faecal	
  samples	
  were	
  collected	
  from	
  healthy	
  adult	
  chickens	
  from	
  two	
  
Ethiopian	
  woredas	
  in	
  May	
  (n=383)	
  and	
  October	
  2011	
  (n=384)	
  	
  
•  Where	
  possible,	
  chickens	
  	
  sampled	
  in	
  May	
  were	
  resampled	
  in	
  October,	
  
giving	
  110	
  further	
  samples	
  
•  McMasters	
  counts	
  were	
  performed	
  on	
  each	
  sample	
  to	
  quanOfy	
  oocysts	
  	
  
[1]	
  
•  A	
  selecOon	
  of	
  samples	
  from	
  those	
  collected	
  in	
  May	
  from	
  each	
  woreda	
  
were	
  speciated	
  using	
  	
  real	
  Ome	
  PCR	
  [2]	
  
Detec2on	
  of	
  Eimeria	
  oocysts	
  in	
  faeces	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
•  427	
  (56%)	
  of	
  samples	
  were	
  posiOve	
  for	
  oocysts,	
  but	
  more	
  samples	
  
were	
  posiOve	
  in	
  October	
  (65%)	
  compared	
  to	
  May	
  (47%)	
  
•  There	
  was	
  moderate	
  clustering	
  of	
  posiOve	
  birds	
  within	
  households,	
  
suggesOng	
  common	
  risk	
  factors	
  or	
  exposure	
  
•  There	
  was	
  no	
  difference	
  in	
  overall	
  prevalence	
  between	
  the	
  regions	
  or	
  
villages,	
  but	
  different	
  species	
  were	
  more	
  prevalent	
  in	
  each	
  region	
  
•  Birds	
  posiOve	
  in	
  May	
  were	
  just	
  as	
  likely	
  to	
  be	
  posiOve	
  in	
  October	
  as	
  
randomly-­‐sampled	
  birds,	
  perhaps	
  suggesOng	
  infecOon	
  with	
  different	
  
species,	
  or	
  immunologically	
  disOnct	
  strains	
  
Region	
   	
  
Mixed	
  species	
  
infec2on	
  
	
  (n	
  =	
  23)	
  
	
  
Single	
  species	
  
infec2on	
  	
  
(n	
  =	
  13)	
  
Species	
   Horro	
   Jarso	
   Fisher’s	
  
exact	
  
	
  p-­‐value	
  
(n	
  =	
  22)	
   (n	
  =	
  25)	
  
E.	
  acervulina	
   9	
  (40%)	
   10	
  (40%)	
   0.8	
   12	
  (52%)	
   1	
  (8%)	
  
E.	
  bruneP	
  1	
   4	
  (18%)	
   5	
  (20%)	
   0.9	
   5	
  (22%)	
   0	
  (0%)	
  
E.	
  maxima	
   5	
  (22%)	
   15	
  (60%)	
   0.022	
   13	
  (57%)	
   4	
  (31%)	
  
E.	
  mi&s	
   0	
  (0%)	
   7	
  (28%)	
   0.012	
   2	
  (9%)	
   2	
  (15%)	
  
E.	
  necatrix	
  1	
   4	
  (18%)	
   2	
  (8%)	
   0.4	
   4	
  (17%)	
   0	
  (0%)	
  
E.	
  praecox	
   21	
  (95%)	
   9	
  (36%)	
   <0.0012	
   17	
  (74%)	
   6	
  (46%)	
  
E.	
  tenella	
  1	
   3	
  (12%)	
   7	
  (28%)	
   0.3	
   3	
  (13%)	
   0	
  (0%)	
  
Frequency	
  of	
  PCR	
  detec2on	
  of	
  Eimeria	
  species	
  in	
  chicken	
  faecal	
  samples	
  
1Considered	
  to	
  be	
  highly	
  pathogenic	
  [3].	
  
2Significantly	
  different	
  (p	
  <0.05).	
  
Conclusions	
  
	
  
•  Eimeria	
  oocysts,	
  including	
  those	
  with	
  the	
  potenOal	
  to	
  be	
  highly	
  pathogenic,	
  
can	
  be	
  frequently	
  detected	
  in	
  faecal	
  samples	
  	
  from	
  village	
  chickens	
  in	
  the	
  
absence	
  of	
  clinical	
  signs	
  of	
  disease	
  
•  There	
  is	
  variaOon	
  in	
  the	
  prevalence	
  of	
  some	
  species	
  between	
  regions,	
  
perhaps	
  suggesOng	
  local	
  risk	
  factors	
  associated	
  with	
  the	
  birds,	
  management	
  
or	
  environment	
  
•  Further	
  invesOgaOons	
  are	
  required	
  to	
  assess	
  the	
  role	
  of	
  Eimeria	
  in	
  clinical	
  
disease	
  and	
  subclinical	
  effects	
  on	
  producOon	
  in	
  village	
  chickens	
  
•  Research	
  is	
  also	
  required	
  as	
  to	
  whether	
  intervenOons	
  may	
  have	
  any	
  posiOve	
  
impacts,	
  or	
  impact	
  negaOvely	
  by	
  disrupOng	
  exisOng	
  endemic	
  stability	
  
Weighing	
  faecal	
  samples	
  
in	
  the	
  Debre	
  Zeit	
  
laboratory	
  in	
  prepara&on	
  
for	
  	
  parasite	
  examina&on	
  
•  All	
  seven	
  species	
  were	
  detected,	
  and	
  highly	
  pathogenic	
  species	
  were	
  detected	
  in	
  
20/47	
  (43%)	
  of	
  samples	
  
•  Mixed	
  infecOons	
  were	
  common	
  (in	
  36	
  samples	
  known	
  to	
  come	
  from	
  a	
  single	
  bird),	
  
and	
  tended	
  to	
  occur	
  with	
  the	
  most	
  prevalent	
  species	
  in	
  each	
  region	
  
	
  

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Prevalence and molecular characterisation of Eimeria species in Ethiopian village chickens

  • 1. Prevalence  and  molecular  characterisa2on  of   Eimeria  species  in  Ethiopian  village  chickens   L.  Luu1  ,  J.  Be*ridge1,2,  R.  Christley1,  K.  Melese3,    D.  Blake4,  T.  Dessie2,  P.  Wigley1  ,  T.  Desta2,5  ,  O.  Hano*e,5,  P.  Kaiser6,  Z.  Terfa2,7,     M.  Collins1  and  S.  Lynch1,2       1  Ins&tute  of  Infec&on  and  Global  Health,  University  of  Liverpool,  UK;  2  Interna&onal  Livestock  Research  Ins&tute,  Addis,  Ababa,  Ethiopia;  3  Debre  Zeit   Agricultural  Research  Centre,  Ethiopian  Ins&tute  for  Agriculture  Research,  Debre  Zeit,  Ethiopia;  4  Department  of  Pathology  and  Infec&ous  Diseases,   Royal  Veterinary  College,  UK;  5  Centre  for  Gene&cs  and  Genomics,  School  of  Biology,  University  of  NoPngham,  UK;  6  The  Roslin  Ins&tute  and  Royal   (Dick)  School  of  Veterinary  Science,  University  of  Edinburgh,  UK;  7  Management  School,  University  of  Liverpool,  UK           Sampling  Strategy                 Eimeria     •  Protozoan  parasites  are  found  worldwide  and    in  all  types  of   chicken  producOon  system   •  7  species  are  known,  but  are  difficult  to  differenOate  based    on   appearance  under  the  microscope   •  Some  species  cause  severe  disease  and  death,  whilst  others    are   only  mildly  pathogenic,  but  may  reduce  growth  rates   References   1.  Permin  A,  Hansen  JW:  Epidemiology,  diagnosis  and  disease  control  of  poultry  parasites.  Food  and  Agriculture  OrganizaOon  of  the  United  NaOons:  Rome;  1998.   2.  Vrba  V,  Blake  DP,  Poplstein  M:  Quan2ta2ve  real-­‐2me  PCR  assays  for  detec2on  and  quan2fica2on  of  all  seven  Eimeria  species  that  infect  the  chicken.  Vet  Parasitol  2010,  174:183–190.   3.  McDougald  LR:  Coccidiosis.  In  Diseases  of  poultry.  11th  ediOon.  Edited  by  Saif  YM,  Barnes  J,  Glisson  JR,  Fadly  AM,  McDougald  LR,  Swayne  DE.  Ames:  Iowa  State  Press  ;  2003.     Roundworm   egg   Eimeria  oocyst   Eimeria  oocysts   X100  magnifica2on   X200  magnifica2on   Methods     •  Faecal  samples  were  collected  from  healthy  adult  chickens  from  two   Ethiopian  woredas  in  May  (n=383)  and  October  2011  (n=384)     •  Where  possible,  chickens    sampled  in  May  were  resampled  in  October,   giving  110  further  samples   •  McMasters  counts  were  performed  on  each  sample  to  quanOfy  oocysts     [1]   •  A  selecOon  of  samples  from  those  collected  in  May  from  each  woreda   were  speciated  using    real  Ome  PCR  [2]   Detec2on  of  Eimeria  oocysts  in  faeces                         •  427  (56%)  of  samples  were  posiOve  for  oocysts,  but  more  samples   were  posiOve  in  October  (65%)  compared  to  May  (47%)   •  There  was  moderate  clustering  of  posiOve  birds  within  households,   suggesOng  common  risk  factors  or  exposure   •  There  was  no  difference  in  overall  prevalence  between  the  regions  or   villages,  but  different  species  were  more  prevalent  in  each  region   •  Birds  posiOve  in  May  were  just  as  likely  to  be  posiOve  in  October  as   randomly-­‐sampled  birds,  perhaps  suggesOng  infecOon  with  different   species,  or  immunologically  disOnct  strains   Region     Mixed  species   infec2on    (n  =  23)     Single  species   infec2on     (n  =  13)   Species   Horro   Jarso   Fisher’s   exact    p-­‐value   (n  =  22)   (n  =  25)   E.  acervulina   9  (40%)   10  (40%)   0.8   12  (52%)   1  (8%)   E.  bruneP  1   4  (18%)   5  (20%)   0.9   5  (22%)   0  (0%)   E.  maxima   5  (22%)   15  (60%)   0.022   13  (57%)   4  (31%)   E.  mi&s   0  (0%)   7  (28%)   0.012   2  (9%)   2  (15%)   E.  necatrix  1   4  (18%)   2  (8%)   0.4   4  (17%)   0  (0%)   E.  praecox   21  (95%)   9  (36%)   <0.0012   17  (74%)   6  (46%)   E.  tenella  1   3  (12%)   7  (28%)   0.3   3  (13%)   0  (0%)   Frequency  of  PCR  detec2on  of  Eimeria  species  in  chicken  faecal  samples   1Considered  to  be  highly  pathogenic  [3].   2Significantly  different  (p  <0.05).   Conclusions     •  Eimeria  oocysts,  including  those  with  the  potenOal  to  be  highly  pathogenic,   can  be  frequently  detected  in  faecal  samples    from  village  chickens  in  the   absence  of  clinical  signs  of  disease   •  There  is  variaOon  in  the  prevalence  of  some  species  between  regions,   perhaps  suggesOng  local  risk  factors  associated  with  the  birds,  management   or  environment   •  Further  invesOgaOons  are  required  to  assess  the  role  of  Eimeria  in  clinical   disease  and  subclinical  effects  on  producOon  in  village  chickens   •  Research  is  also  required  as  to  whether  intervenOons  may  have  any  posiOve   impacts,  or  impact  negaOvely  by  disrupOng  exisOng  endemic  stability   Weighing  faecal  samples   in  the  Debre  Zeit   laboratory  in  prepara&on   for    parasite  examina&on   •  All  seven  species  were  detected,  and  highly  pathogenic  species  were  detected  in   20/47  (43%)  of  samples   •  Mixed  infecOons  were  common  (in  36  samples  known  to  come  from  a  single  bird),   and  tended  to  occur  with  the  most  prevalent  species  in  each  region