Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms

Figures & data

Table 1.  Characterization of R. anatipestifer isolates by seven different methods

Isolates^a	Farm^b/outbreak	Host	Age (days)	Tissue	Method					Antibiotic type^e	Serotype
					Rep-PCR	PFGE^c	Plasmid^d	OmpA	RFLP
10	A/I	Roman geese	50	Joint	Ia	ND^f	I	IA-1	I	TCNCoGFTr	ND
21	A/II	Roman geese	23	Heart	Ia	ND	I	IA-1	I	TOCNGFTr	ND
23A	A/II	Roman geese	23	Liver	Ia	ND	I	IA-1	I	TDCACeNCoB	ND
23B	A/II	Roman geese	23	Lung	Ia	ND	I	IA-1	I	TOCCoF	ND
24A	A/II	Roman geese	23	Liver	Ia	ND	I	IA-1	I	TODCPACeEF	ND
26C	B/III	Roman geese	21	Blood	II	IVA	II	IB-1	II	CPAAoCeNCoFTr	ND
27A	B/III	Roman geese	21	Brain	II	IVB	II	IB-1	II	TOCPAAoCeNECoBFTr	ND
27B	B/III	Roman geese	21	Blood	II	IVB	II	IB-1	II	OAoCeNEGBFEnTr	ND
28B	B/III	Roman geese	21	Brain	II	IVB	II	IB-1	II	CNEGBTr	6
28C	B/III	Roman geese	21	Trachea	II	IVB	II	IB-1	II	CNEBTr	6
29A	B/III	Roman geese	21	Lung	Ia	IIA	III	IA-1	I	TODPACeNECoGF	2
29C	B/III	Roman geese	21	Brain	Ia	IIB	IV	IA-1	I	TODCCoGTr	5
32A	C/IV	Roman geese	14	Blood	Ib	IA-2	IV	IA-1	I	TDCPAAoCeNECoGBr	ND
34A	C/IV	Roman geese	14	Brain	Ib	IA-2	IV	IA-1	I	TODCPAAoCeNECoG	ND
34B	C/IV	Roman geese	14	Bone marrow	Ib	IA-2	IV	IA-1	I	TOCPAAoCeNEGFTr	ND
35A	C/IV	Roman geese	14	Bone marrow	III	III	I	II	III	TOPAAoCeECoGBFTr	4
PDa	E/V	Pekin duck	20	Brain	Ia	VII	IV	IA-1	I	TOCPAAoCeNECoGBFTr	2
PDb	E/V	Pekin duck	20	Bone marrow	Ia	VII	IV	IA-1	I	TODCCeNETr	2
730C	A/VI	Roman geese	22	Brain	Ia	IB	IV	IB-2	II	TOCPNCoGFEnTr	2
807D	A/VI	Roman geese	29	Brain	IV	IA	V	IA-2	I	TCPCeNECoGBFEnTr	6
712	A/VII	Roman geese	29	Brain	IV	VI	VI	IB-1	II	LECoGFTr	ND
725A	D/VIII	Roman geese	28	Trachea	Ia	V	VII	IA-1	I	CAAoCeNCoGBFTr	ND
725B	D/VIII	Roman geese	28	Blood	Ia	V	VII	IA-1	I	TCNCoGFTr	ND
728	D/VIII	Roman geese	31	Brain	Ia	V	VII	IA-1	I	TOCNGFTr	ND
^aArabic number represents R. anatipestifer isolates. The same Arabic number indicates R. anatipestifer isolates isolated from the same goose or duck. Different letters mean R. anatipestifer isolates were isolated from different tissues. PDa is one R. anatipestifer isolate collected from the brain of one Pekin Duck. PDb is another R. anatipestifer isolate collected from the bone marrow of the same Pekin duck.
^bFarm A, Minchun of Chiayi county; Farm B, Mardo of Tainan county; Farm C, Milaun of Yulin county; Farm D, Sihu of Yulin county; and Farm E, Mingen of Nanto county.
^cPFGE of SmaI digested DNA.
^dI, no plasmid; II, 20 kb plasmid; III, 4.5 and 4 kb plasmid; IV, 4 kb plasmid; V, 20 and 2.9 kb plasmid; VI, 10 kb plasmid; VII, 4.5 kb plasmid.
^eT, tetracycline; O, oxytetracycline; D, doxycycline; E, erythromycin; G, gentamicin; C, chloramphenicol; L, lincomycin; P, penicillin; Ao, amoxicillin; A, ampicillin; Ce, cephalothin; B, bacitracin; N, novobiocin; f, flumequine; En, enrofloxacin; Co, colistin; Tr, trimethoprime.
^fND, not done

Figure 1.  Cluster analysis of 24 R. anatipestifer isolates constructed using ClustalV to analyse nucleotide sequences of ompA. Bootstrap values were estimated for this tree using the neighbour-joining model for 1000 replicates. Arabic number, bootstrap value of each cluster.

Figure 2.  Nucleotide sequence alignment and comparison of the ompA of five representative R. anatipestifer isolates. Underlined region, restriction site of restriction enzyme AluI. Arrow, direct repeat region. Repeats II and III encode for PEST regions (Subramaniam et al., 2000), and two are imperfect repeats with a sequence diverse from repeats II and III. –, deletion.

Figure 3.  Rep-PCR patterns of 24 R. anatipestifer isolates. M, 1 kb size marker ranging from 200 bp to 14 kb. N, Negative control (PCR mixture without DNA template).

Figure 4.  PFGE patterns of R. anatipestifer isolates. Genomic DNAs were digested by restriction endonuclease SmaI. M, DNA size marker (48.5-kb phage ladder; BIO-RAD, USA).The same Arabic number means R. anatipestifer isolates were isolated from the same goose or duck. Different letters indicate R. anatipestifer isolates were isolated from different tissues.

Figure 5.  Dendrogram of 24 R. anatipestifer isolates constructed by UPGMA with 3% tolerance to analyse SmaI-digested PFGE pattern. The same Arabic number means R. anatipestifer isolates were isolated from the same goose or duck. Different letters indicate R. anatipestifer isolates were isolated from different tissues.

Subramaniam , S. , Huang , B. , Loh , H. , Kwang , J. , Tan , H.M , Chua , K.L. and Frey , J. 2000 . Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer . Clinical and Diagnostic Laboratory Immunology , 7 : 168 – 174 .

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