AHEAD IN ANIMAL HEALTH
Gallibacterium anatis in Dutch poultry Presence, genetic diversity and association with peritonitis in layers A. Feberwee1, R. Buter1, M. Holstege1, T. Fabri1 1 Royal GD, Deventer, the Netherlands RpoB has been Gallibacterium anatis (GBA) is an opportunistic bacterium (trachea, cloaca and rectum) present in poultry and genotype reported in commercial poultry in and outside Europe. GBA is associated with peritonitis and reduced egg-production often in the presence of other pathogens. Experimentally GBA has shown to be primary pathogenic. High diversity as well as clonal spread of GBA genotypes is reported. This study reports the presence of GBA in Dutch commercial poultry, the genetic diversity and the association with peritonitis in layers. Farm A
Materials and methods Farm D Farm D for 1. Submissions of different poultry types were sampled in the post-mortem (PM) room. Tracheal samples were taken from birds culture (live or fresh dead at arrival in PM room). Farm E Farm D 2. RpoB gene and whole genome sequencing (WGS) analysis was performed on 44 GBA isolates from 33 farms. Also one or more Farm F isolates from farm A to farm E were included (see Figure 1). 3. The association of GBA with peritonitis in layers was evaluated in a Case-Control study. A Case complied with (1) increased mortality, (2) production loss, and (3) peritonitis at PM examination. A Control complied with (1) no disease history, and (2) no Farm dead D lesions at PM. Per Case and Control 15 birds were submitted for PM and from a maximum of 5 individual birds (live or fresh at arrival in PM room) different tissues and bone marrow were sampled aseptically for culture. Mixed effect regression analysis was performed to assess the differences in tissue and bone marrow prevalence. Also WGS was performed of GBA isolates from Farm A Cases and Controls. Farm C Farm B Results Farm E GBA was present in the trachea of different Dutch commercial poultry types (Table 1). RpoB gene and WGS analysis revealed a high genetic diversity of GBA isolates between and within farms (Figure 1). GBA was isolated from tissues in Cases and Controls often in the presence of other bacteria (Table 2). The difference in GBA flock tissue prevalence (Control 1-3: 4/16 (25%) and Case 1-2: 4/6 Farm B (66,7%)) was not statistically significant (p = 0.257, ICC = 0.70 95% CI 0.11-0.98) while the difference in GBA flock prevalence bone marrow (Control 1-3: 0/16 (0%) and Case 1-2: 2/7(28,6%)) was alsmost significant (p = 0.083). WGS showed that different genotypes were present in presence and absence of lesions (Table 3). Farm E Conclusion and discussion Farm B GBA is present in the trachea of different poultry types in Dutch poultry. There is a high genetic variety in GBA isolates which Farm differ F Farm between farms and within farms. WGS is more discriminatory compared to rpoB gene analysis. The association between GBA andF Farm C peritonitis was not confirmed. GBA was also isolated from multiple tissues in birds with lesions and healthy birds with involvement of more than one genotype. Further research has to elucidate the association of GBA with peritonitis in layers.
Acknowledgements This research was funded by the Dutch poultry sector represented by AVINED.
RpoB genotype
Table 1. Presence of GBA in tracheal samples* of Dutch commercial poultry Poultry type Rearing layer reproduction (RLR) Layer reproduction (LR) Rearing commercial layers (RCL) Commercial layers (CL) Rearing meat reproduction (RMR) Meat reproduction (MR) Broilers Total
Submission 1 4 2 39 1 6 11 64
Trachea GBA-positive: n + % 1 (100) 2 (100) 1 (50) 36 (92) 1 (100) 6 (100) 0 (0) 49 (77)
Farm A Farm D Farm D
*Tracheal samples (pool of max. 6 birds): sheep blood agar incubation at max. 48 hours at 34-38oC and 5-10% CO2.
Farm E
Table 2. GBA positive results of Cases and Controls
Farm D Farm F
Flock
Bird Breed no.
PM1
1 2 3 1 2 3 4 1 2 3 4 5 1-5 1-6
Dead Dead Dead5 Live Live Live Live Live Live Live Live Live Live Live
Case 1 Case 1 Case 1 Case 2 Case 2 Case 2 Case 2 Control 1 Control 1 Control 1 Control 1 Control 1 Control 2 Control 3
White White White White White White White Brown Brown Brown Brown Brown Brown Brown
Age (wks)
Bacteriology2,3,4 SPL BM HE 1 1 (3) 1 x 1 (3) x 0 0 1 (3) 0 0 2b 0 2c 1 (2) 0 0 2b 0 0 0 0 0 0 1a 0 0 1 (1) 0 1 (1) 0 0 0 0 0 0 0 0 0 0 0 0
PER 1 x 0 2b 0 0 1b (3) 1a (3) 2a 2a 0 1a 0 0
69 69 69 90 90 90 90 58 58 58 58 58 48 43
LI 1 x 1 (3) 2b 1 (1) 0 0 1 (3) 0 1 (2) 0 0 2a 0
Farm D
Farm A Farm C Farm B Farm E
At arrival; 2PER = peritoneum, SPL = spleen, BM = bone marrow, HE = heart, LI = liver; 30 = no pathogen, 1 = GBA, 1a = GBA + E. coli, 1b = GBA + Enterococcus spp., 2a = Enterococcus spp., 2b = E. coli; 4() = number of colonies for WGS (max. 3); 5Fresh dead at arrival. 1
Farm B
Table 3. Results WGS analysis of GBA isolates per bird in Control 1, and Case 1 and 2 Flock
Bird Breed PM1 Age no. (wks)
PER3 C1 C2
Control 1 Control 1 Case 1 Case 1 Case 1 Case 2 Case 2
1 3 1 2 3 2 4
Brown Brown White White White White White
Live Live Dead Dead Dead2 Live Live
58 58 69 69 69 90 90
4
4
SPL
BM
HE
LI
C3
C1 C2 C3 C1 C2 C3 C1 C2 C3 C1
C2
C3
4 SGT
4 5
4 6
4
Farm E Farm B Farm F Farm F
2 SGT SGT SGT SGT
Farm C
5
5
6 3 3
3 3
3 3 2 8
2 8
10 10 SGT
At arrival; Fresh dead; PER = peritoneum, SPL = spleen, BM = bone marrow, HE = heart, LI = liver, C1/C2/C3 = GBA colony 1-3; 4Genotype is defined when GBA isolates differ a maximum of 100 SNPs (single nucleotide polymorphism); 5Single genotype: no clustering with other isolates. 1
2
3
Figure 1. Results rpoB gene and WGS analysis on 44 GBA isolates. Phylogenetic single SNP analysis using GBA strain UMN179 as reference. Clustering was based on the neighbour joining method (color represents rpoB genotype). DR=duck reproduction, (R)LR=(rearing) layer reproduction, (R)CL = (rearing) commercial layers, MR=meat reproduction.
a.feberwee@gdanimalhealth.com
References Neubauer C. et al., 2009, Avian Pathology.
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