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Veterinary Dermatology 2002, 13, 247 251

Case report

Blackwell Science, Ltd

Isolation and characterization of Pseudomonas aeruginosa from

sheep with fleece rot in northern and middle Jordan

Pseudomonas aeruginosa from fleece rot

SAEB N. EL-SUKHON
Department of Basic Veterinary Medical Sciences, Jordan University of Science & Technology, Irbid, Jordan
(Received 16 February 2001; accepted 13 February 2002)

Abstract A total of 162 sheep fleece samples were collected from 2315 sheep clinically examined for evidence
of dermatitis. The sheep belonged to 32 flocks raised in northern and middle Jordan. Eighty-three samples showed
no obvious abnormalities, whereas the remainder showed exudation (79 samples), fleece discoloration (18) and
fleece roughness (40) and abscesses (7). Seventeen Pseudomonas aeruginosa isolates were obtained from these
samples. Antibacterial resistance in vitro was common; resistance to tetracycline, amoxycillin, erythromycin and
cotrimoxazole was shown by > 90% of the isolates. Resistance to norfloxacin (29.4% of isolates), ciprofloxacin
(17.6%) and amikacin (17.6%) was also demonstrated. Fourteen isolates were serum resistant when assessed after
13 h incubation in sheep and calf sera, and their count increased by 22.9 and 2.53.5 respectively.
Keywords: fleece rot, Pseudomonas aeruginosa, sheep dermatitis.

INTRODUCTION
Wool production is a strategic target of sheep production and the development of dermatitis may reduce the
fleece quality and yield.1,2 Sheep fleece rot is an exudative bacterial dermatitis associated with and favoured
by poor hygienic conditions such as prolonged wetting
of the skin.1,2 Pseudomonas aeruginosa seems to be the
main causative organism in fleece rot and is an important
contributor to the disease process. P. aeruginosa infection
is associated with increased severity of fleece rot and
subsequent flystrike, and the potentially important role
of this organism must be considered in diagnostic
studies and in control strategies such as vaccination.2
Experimental cutaneous inoculation with P. aeruginosa
induced a green discoloration of the fleece of sheep
kept under wet conditions, whereas animals housed
under dry conditions showed no signs of dermatitis.1
This coloration was associated with copious serous
exudation in the fleece rot lesion and with the formation of micro-abscesses.1 Sheep kept in wet conditions,
unlike the dry animals, showed serological reactivity
characterized by an IgG response primarily directed
against outer membrane proteins of the bacterial cell
envelope.1
P. aeruginosa is often resistant to many antibiotics.3,4
In one recent study, 1032% of P. aeruginosa strains were
Correspondence: Saeb N. El-Sukhon, Department of Basic
Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan
University of Science & Technology, PO Box 3030, Irbid, Jordan.
E-mail: sukhon@just.edu.jo
2002 Blackwell Science Ltd

found to be resistant to eight relatively new antibiotics.3

For this reason, a combination of two or more antibiotics
has been advocated in treatment protocols.4,5
The pathogenicity of some P. aeruginosa isolates
may be enhanced by the ability to resist the bactericidal
effect of the complement system; this is termed serum
resistance. Thirty-seven per cent of clinical isolates
in one study were found to be serum resistant.6 Serum
resistance among P. aeruginosa isolates was shown to
vary between serotypes; in one study, all strains of
serotype M were serum sensitive, whereas serotypes A,
B and G were serum resistant.7
In Jordan, no information is available on sheep fleece
rot. The aims of this study were to determine the
prevalence of fleece rot dermatitis among sheep herds
in the northern and middle sectors of Jordan, and
to determine the antibiotic susceptibility and serum
resistance patterns of isolates of P. aeruginosa obtained
from these sheep.

MATERIALS AND METHODS

In total, 162 wool samples were collected from 32 sheep
herds (2315 animals) of both sexes and of different age
groupings. From each herd, 46 samples were collected
aseptically, especially from animals that were showing
skin disease, such as skin /wool discoloration associated
with skin exudation and abscessation.
The samples were processed without delay. They were
cultivated on an array of enrichment and differential
media (Oxoid), including Pseudomonas selective agar,
247

VDE_300.fm Page 248 Friday, September 20, 2002 8:04 PM

248

S. N. El-Sukhon

Table 1. Sheep fleece samples examined for Pseudomonas aeruginosa infection in Jordan
Source/Province
North (Irbid)
North-east (Mafrak)
Middle (Amman)
Middle East (Sahab)
Total

Sex (%)

Age (%)

Clinical signs / No. isolates

No. of
samples

Total No. (%)

of isolates

33
47
55
27
162

16 (48)
23 (49)
25 (45)
15 (55)
79 (49)

17 (52)
24 (51)
30 (55)
12 (45)
83 (51)

7
9
13
6
35

13
22
19
11
65

13
16
23
10
62

4/1
3/2
2/2
5/1
14/6

3
7/1
3
5
18/1

13
11
8
8/1
40/1

3/1

4/2

7/3

10
26/2
38/2
9/2
83/6

2 (6)
5 (10)
6 (11)
4 (15)
17 (10.5)

a, < 6 months; b, 718 months; c, > 24 months.

E, exudate; C, coloration; R, roughness; M, abscesses (mainly), mixed with the other signs; , no obvious signs.

sheep blood agar, MacConkey agar, tryptic soy agar

and tryptic soy broth. The inoculated media were
incubated aerobically at 37 C for 2448 h.
Isolates were characterized biochemically by assessment of oxidase production, citrate utilization, glucose
fermentation, sugar fermentation (fructose, mannitol
and xylose), nitrate reduction, gelatine liquefaction,
urease production, Tween hydrolysis, by growth at low
(5 C) and high (42 C) temperatures, and by motility
according to the methods of Koneman et al.8 and
Cowan and Steel.9 The antibiotic susceptibility of
the isolates was studied by determining the minimum
inhibitory concentration (MIC) in solid medium
according to the guidelines of the NCCLS.10
Blood from six healthy sheep and three healthy
calves was collected, the sera of each species pooled,
and held at 22 C for future use. A serum resistance
assay was performed using these sera, as described
previously.11 In brief, the sera were thawed at room
temperature and an aliquot heated at 56 C for 30 min
in order to inactivate the complement components.
An 18-h-old culture of each isolate, grown in tryptic
soy agar, was subcultured into a tryptic soy broth. Cells
in the exponential growth phase were harvested and
incubated ( 3 107/mL) in heat-treated and untreated
sera at 37 C for 3 h. The viable bacterial count was
determined by plating serial 10-fold dilutions of the
serum onto tryptic soy agar in triplicate.11

RESULTS
A total of 162 fleece samples were examined. The
numbers of samples from males and females were
comparable and most were collected from sheep 12
years of age (Table 1). Different clinical signs were
observed in 79 of the examined sheep and the remaining
83 sheep showed signs of pruritus on observation but
no additional abnormalities upon physical examination
(Table 1). Exudation was recorded in 14 (6.5%) sheep,
greenish/yellowish discoloration in 18 (11%), fleece
roughness in 40 (24.7%) and abcessation mixed with
other clinical signs was recorded in 7 (4%) (Table 1).
P. aeruginosa was isolated from 17 sheep (10.5%).
Eleven isolates were from animals with clinical signs
and the remaining six were from animals showing only
pruritus (Table 1). The majority of isolates (13 of 17)
were recovered between December and April.
The antibiotic resistance patterns of the isolates
against 16 antibiotics that are commonly used in Jordan
are shown in Table 2. The relatively new antibiotics
like norfloxacin, ciprofloxacin and amikacin were
not fully effective with 29.4, 17.6 and 17.6% of strains,
respectively, showing resistance in vitro. Antibiotics
used in the field for longer periods, including tetracycline, amoxycillin, erythromycin and cotrimoxazole
were ineffective against > 90% of the isolates. A
number of isolates showed intermediate resistance
to norfloxacin, ciprofloxacin, amikacin, enrofloxacin,

Figure 1. Changes in microbial counts (log10)

of 17 Pseudomonas aeruginosa isolates
obtained from sheep fleece rot cases in
Jordan following incubation with untreated
and heat-inactivated sheep serum.
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 247 251

VDE_300.fm Page 249 Friday, September 20, 2002 8:04 PM

Table 2. Antibiotic susceptibility of 17 Pseudomonas aeruginosa isolates from cases of fleece rot in Jordanian sheep

Gentamicin
Norfloxacin
Ciprofloxacin
Amikacin
Enrofloxacin
Tetracycline
Amoxicillin
Erythromycin
Ampicillin
Kanamycin
Streptomycin
Co-trimoxazol
Neomycin
Doxicyclin
Nalidixic acid
Nitrofuantoin

10
10
5
30
5
30
35
25
10
30
10
25
30
30
30
300

Sensitivity range (mm)

Inhibition zone (mm) measured from the edge of the antibiotic disc of 17 strains of P. aeruginosa

10

11

12

13

14

15

16

17

% R*

12
15
15
14
15
14
22
13
22
13
11
11
12
12
13
14

13 14
16 20
16 20
15 17
16 20
15 18
23 30
14 22
2330
14 17
1214
1216
1316
1315
14 18
1516

15
21
21
18
21
19
30
23
31
18
15
17
17
16
19
17

17
21
22
17
13
10
19
9
13
11
10
10
10
17
13
10

11
12
22
18
18
10
19
9
13
11
9
10
10
14
14
10

9
12
24
18
19
12
22
9
14
10
10
10
10
14
19
18

9
18
13
21
21
11
22
10
13
12
15
10
12
13
14
12

12
19
21
22
15
10
21
11
15
13
11
10
10
11
12
17

15
17
15
22
14
10
20
10
10
13
12
11
9
11
21
12

17
22
17
21
12
12
21
10
14
14
16
10
11
10
11
14

15
22
16
25
13
11
24
12
25
11
12
11
11
9
12
14

15
24
21
24
17
9
17
15
14
10
11
17
12
10
22
13

10
18
22
24
15
8
19
12
14
14
11
10
13
10
13
10

17
15
17
12
21
10
19
9
15
15
15
10
12
18
14
11

12
21
12
23
14
12
17
10
14
16
12
9
19
12
16
19

16
22
21
24
10
15
16
13
12
18
10
8
11
17
13
12

18
14
17
11
15
11
20
10
23
18
10
8
18
11
21
13

10
16
16
14
22
12
21
11
10
14
10
8
14
19
15
17

19
17
18
24
19
10
20
10
23
11
11
9
17
17
15
15

15
15
18
25
18
9
20
9
16
11
11
10
13
11
16
15

41
29.4
17.6
17.6
53
94
94
94
88
59
65
94
65
53
35.3
70

Pseudomonas aeruginosa from fleece rot

R, resistant; I, intermediate susceptibility; S, sensitive.

Numbers in bold represent resistant results.
*Per cent resistant.

249

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 247251

Antibiotic

Conc.
g/disc

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250

S. N. El-Sukhon

Figure 2. Changes in microbial counts

(log10) of 17 Pseudomonas aeruginosa isolates
obtained from sheep fleece rot cases in
Jordan following incubation with untreated
and heat-inactivated calf serum.

tetracyclin, amoxycillin, erythromycin, ampicillin,

kanamycin, streptomycin, neomycin, doxicyclin, nalidixic
acid and nitrofuantoin (Table 2).
The serum resistance patterns of the isolates of
P. aeruginosa are shown in Figs 1 and 2. Three of the 17
isolates were recorded as serum sensitive as their
counts (expressed as log10) decreased by 0.71.3 when
incubated with untreated sheep serum (Fig. 1). Counts
for the remaining 14 of these isolates increased by
2.02.9 when incubated in untreated sheep serum,
and colony counts of all 17 isolates increased by 23.1
when incubated in the heat-inactivated sheep serum.
A similar pattern was seen with calf serum with same
three strains being sensitive to untreated serum,
although colony counts in the serum resistant isolates
were slightly higher, ranging from 2.5 to 3.5.

DISCUSSION
This survey indicates that sheep fleece rot is prevalent
in Jordan and that P. aeruginosa may be isolated from
a proportion of affected flocks. Vaccination against
this disease is not currently carried out but could be of
value in future control strategies.
The majority of isolates (13 of 17) were recovered
between December and April, which is the wet/humid
season in Jordan. During March and April, the climate
becomes warmer and the fly population increases. This
supports previous observations on the importance
of moisture in disease susceptibility.1,2 The role of flies
in the transmission of this disease2 in Jordan requires
further assessment. Isolation of P. aeruginosa from adult
sheep suggests that age may also be involved in disease
susceptibility.
P. aeruginosa is well-known for its frequent resistance to a wide spectrum of antibiotics. For example,
in the UK, 18.5% of P. aeruginosa isolates obtained
from humans were resistant to, or had reduced susceptibility to, recently introduced beta-lactam antibiotics.12
In Jordan, antibiotics, including fluoroquinolones,
are very commonly used in sheep production without
veterinary supervision and control. This misuse of
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 247 251

antibiotics may be reflected by and favour the high

level of resistance to antibiotics observed among the
P. aeruginosa isolates studied here. Consequently, there
is a need for better control of antibiotic usage in
Jordan, and bacterial culture and sensitivity testing
may be advised to assist antibiotic selection in the
treatment of established Pseudomonas infections.
The bactericidal effect of serum may reflect the
activity of complement components. Although 14
isolates revealed the same serum resistance patterns
toward the sera from both ovine and bovine sources,
their count was higher when incubated in the calf
serum than in sheep serum, whereas three (18%) serumsensitive isolates revealed a lower count in the calf
serum. The variability of the cidal potency of the
bovine and ovine serum may reflect a degree of in vivo
adaptation of the isolates to the host species.
This is the first study of ovine fleece rot in Jordan.
Further investigations into the factors which favour
this disease are required. Antimicrobial resistance is
common among P. aeruginosa isolates obtained from
affected sheep and antibiotic usage should be controlled. Sheep migration and movements between Jordan
and neighbouring countries should be controlled and
quarantine measures respected to limit the spread of
the disease.

REFERENCES
1. Chin, J.C., Dai, Y., Watts, J.E. Antibody-response
against Pseudomonas aeruginosa membrane-proteins in
experimentally infected sheep. Veterinary Microbiology,
1995; 43: 2132.
2. Kingsford, N.M., Raadsma, H.W. The occurrence of
Pseudomonas aeruginosa in fleece washings from sheep
affected and unaffected with fleece rot. Veterinary
Microbiology, 1997; 54: 27585.
3. Bonfiglio, G., Carciotto, V., Russo, G. et al. Antibiotic
resistance in Pseudomonas aeruginosa, an Italian survey.
Journal of Antimicrobial Agents and Chemotherapy, 1998;
41: 30710.
4. Eaglesome, M.D., Garcia, M.M. Comparisons of antibiotic combinations to control Pseudomonas aeruginosa

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in bovine semen. Canadian Journal of Veterinary Research

Review, 1995; 59: 73 5.
Gerceker, A.A., Gurler, B. In vitro activities of various
antibiotics, alone and in combination with amikacin
against Pseudomonas aeruginosa. Journal of Antimicrobial Agents and Chemotherapy, 1995; 36: 70711.
Pajdak, E., Szkarlat, A., Kobylarz, K. Characteristics of
clinical isolates of Pseudomonas aeruginosa. I. Resistance
to serum bactericidal effect and production of proteolytic enzymes. Archivumimmunologiae et Therapiae
Experimentalis (Wars.), 1990; 38: 30914.
Nippon, S. Study on serum-sensitivity of Pseudomonas
aeruginosa. Gakkai Zasshi, 1991; 65: 90917.
Koneman, E.W., Allen, S.D., Dowell, Jr. V.R., et al. Color
Atlas and Textbook of Diagnostic Microbiology, 2nd edn.
Philadelphia: Lippincott, 1983: 12585.

251

9. Cowan, S.T. Cowan and Steels Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge: Cambridge University Press, 1975: 903.
10. National Committee for Clinical Laboratory Standards.
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Avenue, Villanova, PA 19085.
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resistance by defined lipopolysaccharide mutants and an
acapsular mutant of uropathogenic Escherichia coli O75:
K5. Infection and Immunity, 1998; 66: 424453.
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3009.

Rsum 162 prlvements de selles ont t obtenus partir de 2315 moutons examins pour recherche de
dermatite. Les moutons appartenaient 32 troupeaux levs dans le nord et le centre de la Jordanie. 83 prlvements nont pas montr danomalie, alors que les autres prsentaient une exudation (79 prlvements), une
dcoloration (18) et une duret (40). 17 isolements de Pseudomonas aeruginosa ont t obtenus de ces prlvements. Une rsistance aux antibiotiques a t frquemment observe; Plus de 90% des souches taient rsistantes
la ttracycline, lamoxycilline, lrythromycine et le co-trimoxazole. Une rsistance la norfloxacine (29.4%),
la ciprofloxacine (17.6%) et lamikacine (17.6%) a galement t observe. 14 souches taient rsistantes aprs
incubation d1 3 heures dans le serum de mouton et de vache respectivement.
Resumen Se recogieron un total de 162 muestras del velln de 2315 ovejas examinadas clnicamente de dermatitis.
Las ovejas pertenecan a 32 rebaos criados en la zona norte y centro de Jordania. Ochenta y tres muestras no
mostraron anomalas evidentes mientras que el resto mostraban exudacin (79 muestras), decoloracin del velln
(18) y aspereza (40). Se obtuvieron diecisiete aislamientos de Pseudomonas aeruginosa en estas muestras. Fueron
frecuentes las resistencias antibacterianas in vitro; en ms del 90% de los aislamientos se observ resistencia a la
tetraciclina, amoxicilina, eritromicina y co-trimoxazol. Tambin se demostr resistencia a la norfloxacina (29.4%
de los aislamientos), ciprofloxacina (17.6%) y amicacina (17.6%). Catorce aislamientos eran suero-resistentes al
probarse despus de 13 horas de incubacin en sueros de oveja y ternera, respectivamente.
Zusammenfassung Insgesamt 162 Vliesproben wurden von 2315 Schafen gesammelt, die klinisch auf Anzeichen
von Dermatitis untersucht wurden. Die Schafe stammten von 32 Herden im nrdlichen und mittleren Jordanien.
Dreiundachtzig Proben zeigten keine Aufflligkeiten. Die brigen zeigten Exudation (79 Proben), Verfrbung
(18) und Rauheit (40) des Vlies. Aus diesen Proben wurden 17 Pseudomonas aeruginosa Isolate gewonnen.
Antibakterielle Resistenzen in vitro waren hufig. Mehr als 90% der Isolate zeigten eine Resistenz gegenber
Tetrazyklin, Amoxicillin, Erythromycin und Cotrimoxazol. Resistenzen gegenber Norfloxacin (29,4% der
Proben), Ciprofloxacin (17,6%) und Amikacin (17,6%) wurden ebenfalls nachgewiesen. Nach ein- bis dreistndiger
Inkubation in Schaf- und Klberserum stellten sich vierzehn Isolate als resistent gegenber Serum heraus.

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 247251