J Vet Diagn Invest 15:26–29 (2003)

Antimicrobial susceptibility of Riemerella anatipestifer isolated

from ducks and the efficacy of ceftiofur treatment
Chao-Fu Chang, Wen-Hwa Lin, Tung-Mao Yeh, Tai-Sheng Chiang, Yung-Fu Chang

Abstract. The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur
and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum
inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 mg/ml;
ceftiofur was 32 mg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 mg/ml; nalidixic
acid, nitrofurantoin, and sulfamethoxazole were 128 mg/ml; amikacin, ampicillin, gentamicin, lincomycin, spec-
tinomycin, streptomycin, tetracycline, and trimethoprim were $256 mg/ml. The therapeutic efficacy of ceftiofur
against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental
ducks were infected through the infraorbital sinus with 1 ml of 9 3 109 CFU of R. anatipestifer. Ceftiofur (0,
0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg
resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.

Riemerella anatipestifer is a major bacterial patho- Materials and methods

gen of ducks in Taiwan and other countries causing a
disease variously known as infectious serositis, new
duck disease, duck septicemia, or anatipestifer septi-
cemia.3,19 This serious and widespread disease causes
major economic losses in the duck industry through
high mortality, reduced growth rate, poor feed con-
version, increased condemnations, and high treatment
costs.3
The occurrence of different serotypes of R. anati-
pestifer in field cases has been reported.2,3,5,7,18 Unfor-
tunately, no cross-protection has been observed with
inactivated bacterins made from different serotypes of
R. anatipestifer.16 This lack of cross-protection seri-
ously limits the usefulness of immunization in con-
trolling this disease. Consequently, chemotherapy is
very important in the treatment of ducks infected with
R. anatipestifer.
The availability of ceftiofur, a third-generation ceph-
alosporin antibiotic, which is active against many
gram-positive and gram-negative bacteria, provided
the impetus for a study in which ceftiofur and 16 other
antimicrobial agents were compared for their potential
usefulness in controlling R. anatipestifer infections in
ducks.11 The in vitro minimum inhibitory concentra-
tions (MICs) of 17 antimicrobial agents against field
isolates of R. anatipestifer were determined. The ther-
apeutic efficacy of ceftiofur against R. anatipestifer in-
fection was determined in ducks inoculated with a vir-
ulent strain of the organism.
From the Department of Veterinary Medicine, National Taiwan
University, 142 ZhouShan Road, Taipei 106, Taiwan (C.-F. Chang,
Lin, Yeh, Chiang), and the Department of Population Medicine and
Diagnostic Science, College of Veterinary Medicine, Cornell Uni-
versity, Ithaca, NY 14853 (Y.-F. Chang).
26
Bacterial strains. Fifty strains of R. anatipestifer were
chosen, all isolated from ducks with infectious serositis, and
serotyped at the Laboratory of Clinical Microbiology, De-
partment of Veterinary Medicine, National Taiwan Univer-
sity. American Type Culture Collection (ATCC) quality con-
trol strains, Escherichia coli ATCC 25922, Enterococcus
faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC
27853, were used in this study.6,8 The test strains were stored
at 270 C before testing.
In vitro susceptibility tests. Agar-dilution MIC tests were
conducted as previously described.6,8 The following antimi-
crobial agents were tested: amikacin, ampicillin, ceftiofur
sodium, cephalothin, chloramphenicol, flumequine, genta-
micin, kanamycin, lincomycin, nalidixic acid, nitrofurantoin,
penicillin, spectinomycin, streptomycin, sulfamethoxazole,
tetracycline, and trimethoprim. With the exception of cef-
tiofur, which was provided by the Pharmacia & Upjohn
Pharmaceutical Company,a all other antimicrobials were pur-
chased from Sigma.b The dilution ranges used for these an-
timicrobial agents were 0.03–256 mg/ml, except sulfamthox-
azole with 0.5–512 mg/ml. Inoculum was dispensed, with a
replicatorc housing 3-mm pins.
Selection and preparation of the most virulent strain for
infection. A total of forty-eight, 4-week-old, 600–700 g
ducks were used to determinate the virulence of the 7 dif-
ferent R. anatipestifer serotypes. The ducks were distributed
randomly into 7 treatment groups and 1 control group.
Among them, 1 group served as control. There were 3 male
and 3 female ducks in each group. The ducks received drink-
ing water and antibiotic-free commercial feed ad libitum.
The most virulent R. anatipestifer strain, as judged by duck
mortality, was grown on tryptic soy agard supplemented with
sheep blood (5%, v/v) for 18 hr at 37 C. The cells were
harvested by scraping and suspending them in 0.85% sterile
saline. A suspension containing 9 3 109 CFU/ml, as deter-
mined by plate count, was used to infect the experimental
ducks.
 MICs of antimicrobial agents against Riemerella anatipestifer and efficacy of ceftiofur in ducks 27

Table 1. In vitro activities of various antimicrobial agents against Riemerella anatipestifer isolated from ducks in Taiwan (n 5 50).

Cumulative (%) MIC (mg/ml)

Antimicrobial
agent 0.12 0.25 0.5 1 2 4 8 16 32 64 128

Amikacin 6(12) 18(40) 26(52)

Ampicillin 4(8) 8(16) 10(20) 21(42) 26(52) 32(64) 37(74)
Ceftiofur 4(8) 8(16) 28(56) 30(60) 33(66) 34(68) 44(88) 50(100)
Cephalothin 4(8) 18(36) 20(40) 24(48) 30(60) 37(74) 48(96) 50(100)
Chloramphenicol 8(16) 19(38) 22(44) 25(50) 31(62) 35(70) 47(94) 50(100)
Flumequine 6(12) 18(36) 20(40) 26(52) 37(74) 38(76) 45(90) 50(100)
Gentamicin 6(12) 9(18) 12(24) 20(40) 27(54) 37(74)
Kanamycin 3(6) 5(10) 7(14) 29(58) 45(90) 50(100)
Licomycin 10(20) 29(58)
Nalidixic acid 1(2) 8(16) 13(26) 20(40) 27(54) 38(76) 50(100)
Nitrofurantoin 5(10) 12(24) 19(38) 28(56) 39(78) 42(84) 50(100)
Penicillin 3(6) 4(8) 20(40) 24(48) 28(56) 34(68) 50(100)
Spectinomycin 4(8) 18(36) 20(40) 28(56) 37(74)
Streptomycin 7(14) 14(28) 20(40) 26(52) 40(80)
Sulfamethoxazole 3(6) 18(36) 27(54) 50(100)
Tetracycline 8(16) 13(26) 17(34) 21(42) 22(44) 26(52) 37(74)
Trimethoprim 12(24) 23(46) 39(78)

In vivo ceftiofur efficacy. Four groups of ducks (30 ducks Results

per group) were treated with various doses of ceftiofur (0.25,
0.5, 1.0, and 2.0 mg/kg body weight, respectively). Controls Because interpretive criteria with R. anatipestifer
(10 ducks per group) were either untreated or injected with isolated from ducks are not available for all antimicro-
saline but not challenged with R. anatipestifer. Except for bial agents tested, isolates in the present study should
the saline control group, all ducks were infected by injecting not be classified as susceptible or resistant; therefore,
1 ml of saline containing 9 3 109 CFU of the highly virulent only MIC data are presented. The in vitro activities of
isolate of R. anatipestifer into the infraorbital sinus. Ceftiof- various antimicrobials against isolates of R. anatipes-
ur was dissolved in sterile deionized water and injected sub- tifer are listed in Table 1, and the MICs are provided
cutaneously into the neck of each duck 5 hr after infection.
in Table 2. The 5 most potent antibacterials based on
Mortality was recorded daily for 2 wk after treatment. Ducks
that died were necropsied and the heart, liver, lung, brain, MIC90 values in descending order were penicillin (16
and air sac were cultured on tryptic soy agard containing 5% mg/ml), ceftiofur (32 mg/ml), cephalothin (64 mg/ml),
sheep red blood cells for the isolation of R. anatipestifer. chloramphenicol (64 mg/ml), flumequine (64 mg/ml),
All ducks surviving at the end of the experiment were sac- and kanamycin (64 mg/ml each). Nalidixic acid, nitro-
rificed and examined for pathological lesions. furantoin, and sulfamethoxazole had MIC90 values of
128 mg/ml. The MIC90 values of amikacin, ampicillin,
Table 2. MIC50, MIC70, and MIC90 for Riemerella anatipestifer gentamicin, lincomycin, spectinomycin, streptomycin,
isolated from ducks in Taiwan (n 5 50). tetracycline, and trimethoprim were $256 mg/ml.
Antimicrobial Range MIC50 MIC70 MIC90
On the basis of MIC50 values, the 4 most potent
agent (mg/ml) (mg/ml) (mg/ml) (mg/ml) antimicrobials and their rank order were the same as
based on MIC90 values, although their concentration
Amikacin 32–.256 128 256 .256
Ampicillin 2–.256 32 128 .256 values were reduced with penicillin (2 mg/ml), cef-
Ceftiofur 0.12–.32 4 8 32 tiofur (4 mg/ml), chloramphenicol (8 mg/ml), and flu-
Cephalothin 1–128 16 32 64 mequine (8 mg/ml). The MIC50 of cephalothin and ni-
Chloramphenicol 1–128 8 32 64 trofurantoin was 16 mg/ml; ampicillin, kanamycin, nal-
Flumequine 1–128 8 16 64
Gentamicin 4–.256 64 128 .256
idixic acid, and spectinomycin was 32 mg/ml; genta-
Kanamycin 4–128 32 64 64 micin streptomycin, sulfamethoxazole, and
Licomycin 64–.256 128 256 .256 tetracycline was 64 mg/ml; the remaining antimicro-
Nalidixic acid 2–128 32 64 128 bials was 128 mg/ml. The MIC70 of ceftiofur was 8 mg/
Nitrofurantoin 2–128 16 32 128 ml; flumequine and penicillin was 16 mg/ml; cepha-
Penicillin 0.12–16 2 16 16
Spectinomycin 4–256 32 128 256 lothin, chloramphenicol, and nitrofurantoin was 32 mg/
Streptomycin 8–256 64 128 256 ml; kanamycin and nalidixic acid was 64 mg/ml; am-
Sulfamethoxazole 16–128 64 128 128 picillin, gentamicin, spectinomycin, streptomycin,
Tetracycline 8–256 64 128 256 sulfamethoxazole, tetracycline, and trimethoprim was
Trimethoprim 32–.256 128 128 .256
128 mg/ml; amikacin and licomycin had the highest
28
Table 3. Duck mortality associated with various Riemerella an-
atipestifer serotypes.
Serotype Mortality*
1 6/6
2 0/6
3 3/6
5 2/6
8 0/6
9 4/6
10 0/6
Control 0/6
* Dead/inoculated.
value of 256 mg/ml. Amikacin, lincomycin, and tri-
methoprim showed the highest MIC50, MIC70, and
MIC90 values as compared with other antimicrobials
(Table 2).
Serotyping of the isolates yielded 7 different sero-
types that were tested for virulence using mortality as
the criterion (Table 3). A representative isolate of each
serotype was chosen at random for testing. The rep-
resentative of serotype 1, strain number RA-45, which
caused 100% mortality (Table 3), was clearly the most
virulent and was used as the challenge strain in effi-
cacy test.
Ceftiofur, given as a single subcutaneous dose 5
hours after an infraorbital challenge with the RA-45,
reduced mortality in a dose-related fashion, from 90%
in the untreated controls to about 27% in ducks re-
ceiving 2 mg/kg (Table 4). Similarly, the median time
of death of the ducks more than doubled from 2.0 days
in the untreated control group to 4.5 days in the groups
receiving 1.0 or 2.0 mg/kg of ceftiofur. It should be
noted that the MIC value of this organism was 0.5 mg/
ml that was less than the susceptible MIC breakpoint,
2 mg/ml, of cattle and swine pathogen.8
The ducks infected experimentally with R. anati-
pestifer serotype-1 exhibited signs of listlessness, oc-
ular discharge, and swollen eyelids at 5 hours after
challenge. Ducks that died from R. anatipestifer infec-
tion had macroscopic lesions of pericarditis, perihep-
atitis, meningitis, and fibrinous airsacculitis. Riemer-
ella anatipestifer was isolated from all lesions as well
as from the brain. Some of the surviving ducks that
received lower doses of ceftiofur were emaciated and
showed incoordination, shaking of the head, torticollis,
and loss of righting reflex, suggesting severe neural
signs. However, none of these signs was observed in
surviving ducks that had received ceftiofur at the high-
est dose (2 mg/kg) given.
Discussion
The in vitro susceptibility of veterinary pathogens
to antimicrobials can provide valuable guidance in the
choice of chemotherapy. Only a limited amount of data
Chang et al.
Table 4. Mortality and median time of death in ducks treated
with various dosages of ceftiofur after challenge with Riemerella
anatipestifer.
Dose Median time
(mg/kg) Mortality* of death (day)
0 9/10 2.0
0.25 23/30 3.0
0.5 24/30 3.5
1.0 14/30 4.5
2.0 8/30 4.5
Challenged with saline 0/10 —
* Mortality is expressed as dead/tested.
is available on the MICs of antimicrobial agents
against R. anatipestifer.1 A previous report showed
that all 5 strains of R. anatipestifer, isolated from
ducks in the United States, grew on Mueller-Hinton
containing kanamycin at a concentration of 532.8 mg/
ml.1 In contrast, all isolates were inhibited by kana-
mycin at a concentration of 128 mg/ml in this study.
The difference in antimicrobial activity against isolates
from the 2 countries could be attributed to many fac-
tors.20 Variations in antimicrobial agent usage from one
country to another could be a reason. Another factor
could be differences in the serotypes of an organism
from one country to another. Previous report showed
differences in antibiograms on the basis of the sero-
types of US Streptococcus suis isolates.12 There are no
authentic MIC values of antimicrobial agents against
R. anatipestifer other than kanamycin available to date,
so no comparison can be addressed further.
The use of induced disease models in evaluating the
efficacy of antimicrobials in ducks has been docu-
mented. The use of flumequine for the control of mor-
tality in experimental R. anatipestifer infections13 as
well as the reduction of mortality in R. anatipestifer–
infected ducks by the application of lincomycin and
spectinomycin, penicillin and streptomycin, oxytetra-
cycline, and spectinomycin, respectively, has been re-
ported.17 The R. anatipestifer isolates used in the cur-
rent study were composed of 7 serotypes, 3 of which
did not cause mortality when single isolates chosen
randomly were injected into ducks. This finding ne-
cessitated a preliminary experiment to find a lethal iso-
late because mortality was the criterion for evaluating
efficacy of an antibacterial. The serotype-1 isolate that
caused 100% mortality in the preliminary experiment
and was susceptible to ceftiofur appeared ideal for
such experimental purposes.
Ceftiofur, a broad-spectrum cephalosporin antibiotic
that had not been previously tested in ducks, reduced
mortality from 90 to 27% and more than doubled the
median time to death when administered at 2.0 mg/kg
in the experimental model. In addition, the survivors
at this dose level did not exhibit any of the adverse
 MICs of antimicrobial agents against Riemerella anatipestifer and efficacy of ceftiofur in ducks
signs characteristic of infectious serositis, which were
observed in survivors or groups receiving lower doses.
The findings of the present study imply that ceftiofur
is as useful as other antimicrobials in the practical con-
trol of bacterial infections common to the duck indus-
try, despite development of resistance factors. Al-
though the most convenient method of administration
of antimicrobials to ducks is through the feed, paren-
teral administration of ceftiofur was studied as a means
of treating sick birds individually.
Antimicrobial resistance can be plasmid, chromo-
some, or transposon mediated (or all) in bacteria.15 Two
plasmids from R. anatipestifer have been previously
characterized, both carried a virulence-associated gene
but no antibiotic resistant genes.4,21 This indicates that
the antimicrobial resistance in R. anatipestifer is prob-
ably chromosome mediated. The genetic mechanism of
chromosome-mediated antimicrobial resistance in R.
anatipestifer is unknown. Studies of other bacteria, in-
cluding E. coli, P. aeruginosa, Nesseria gonorrhoeae,
and Haemophilus influenzae, have indicated that there
are multidrug resistance (MDR) efflux systems.9,10,14
The MDR efflux system has been classified into 4 trans-
porter families; the ATP-binding cassette superfamily,
the major facilitator superfamily, the small drug resis-
tance family, and the resistance–nodulation–cell divi-
sion (RND) family.14 It is known that RND family
transporters can actively export a wide variety of anti-
biotics in gram-negative organisms.9 Determination of
whether or not the antibiotic resistance in R. anatipes-
tifer belongs to this family awaits further studies.
Acknowledgements
We are grateful to Dr. Sean P. McDonough for helpful
discussion and editorial assistance. This work was support
by a grant (NSC83-0409-B-002-141) from National Science
Council of Republic of China in Taiwan.
Sources and manufacturers
a. Pharmacia & Upjohn, Kalamazoo, MI.
b. Sigma Chemical Co., St. Louis, MO.
c. Cathra, Automad, MN.
d. Difco, Detroit, MI.
References
1. Bangun A, Tripathy DN, Hanson LE: 1981, Studies of Pasteu-
rella anatipestifer: an approach to its classification. Avian Dis
25:326–337.
2. Bisgaard M: 1982, Antigenic studies on Pasteurella anatipes-
tifer species incertae sedis, using slide and tube agglutination.
Avian Pathol 11:341–350.
29
3. Chang CF: 1984, Pathogenesis of Pasteurella anatipestifer in-
fection in ducks and their sensitivity to antibacterial agents. Tai-
wan J Vet Med Anim Hus 43:40–46.
4. Chang CF, Hung PE, Chang YF: 1998, Molecular characteriza-
tion of a plasmid isolated from Riemerella anatipestifer. Avian
Pathol 27:339–345.
5. Harry EG: 1969, Pasteurella (Pfeifferella) anatipestifer sero-
types isolated from cases of anatipestifer septicaemia in ducks.
Vet Rec 84:673.
6. Jorgensen JH, Turnidge JD, Washington JA: 1999, Antimicro-
bial susceptibility tests: dilution and agar diffusion methods. In:
Manual of clinical microbiology, ed. Murray PR, 7th ed., pp.
1526–1543. ASM Press, Washington, DC.
7. Loh H, Teo TP, Tan HC: 1992, Serotypes of Pasteurella ana-
tipestifer isolates from ducks in Singapore: a proposal of new
serotypes. Avian Pathol 21:453–459.
8. National Committee for Clinical Laboratory Standards
(NCCLS): 1999, Performance standards for antimicrobial disk
and dilution susceptibility tests for bacteria isolated from ani-
mals, tentative standard, M31-A. NCCLS, Wayne, PA.
9. Nikaido H: 1998, Antibiotic resistance caused by gram-negative
multidrug efflux pumps. Clin Infect Dis 27S-1:s32–s41.
10. Paulsen IT, Skurray RA, Tam R, et al.: 1996, The SMR family:
a novel family of multidrug efflux proteins involved with the
efflux of lipophilic drugs. Mol Microbiol 19:1167–1175.
11. Plumb DC: 1999, Veterinary drug handbook, 3rd ed., pp. 110–
116. Iowa State University Press. Ames, IA.
12. Reams RY, Glickman LT, Harrington DD, et al.: 1993, Strep-
tococcus suis infection in swine: a retrospective study of 256
cases. Part I. Epidemiologic factors and antibiotic susceptibility
patterns. J Vet Diagn Investig 5:363–367.
13. Salem B: 1991, Trials for controlling Pasteurella anatipestifer
infection in ducks. Avian Dis 26:276–282.
14. Saier MH, Paulsen IT, Sliwinski MK, et al.: 1998, Evolutionary
origins of multidrug and drug-specific efflux pumps in bacteria.
FASEB J 12:265–274.
15. Salyers AA, Whitt DD: 1994, Antibiotics: mechanism of action
and mechanisms of bacterial resistance. In: Bacterial pathogen-
esis: a molecular approach, pp. 97–110. ASM Press, Washing-
ton, DC.
16. Sandhu TS: 1979, Immunization of White Pekin ducklings
against Pasteurella anatipestifer infection. Avian Dis 23:662–
669.
17. Sandhu TS, Dean WF: 1980, Effect of chemotherapeutic agents
on Pasteurella anatipestifer infection in white Pekin ducklings.
Poultry Sci 59:1027–1030.
18. Sandhu T, Harry EG: 1981, Serotypes of Pasteurella anatipes-
tifer from commercial White Pekin ducks in the United States.
Avian Dis 25:497–502.
19. Sandhu TS, Rimler RB: 1997, Riemerella anatipestifer infec-
tion. In: Diseases of poultry, ed. Calnek BW, 10th ed., pp. 161–
166. Iowa State University Press, Ames, IA.
20. Singh R, Teo TP, Tay YH, et al.: 1989, Biochemical character-
istics and drug sensitivity of Pasteurella anatipestifer isolates
from Singapore ducks. Singap J Prim Ind 17:59–62.
21. Weng SC, Lin WH, Chang YF, et al.: 1999, Identification of a
novel virulence-associated protein gene and an insertion se-
quence element ISRa1 in a plasmid of Riemerella anatipestifer.
FEMS Microbiol Lett 179:11–19.