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Journal of Animal Science Advances
Isolation, Histopathological and molecular detection of
Yesinia Pseudotuberclosis infection in Sheep in Turkey.
Neval Berrin Arserim, Sevgi Kalkanli, Hasan İçen, Nurettin Işık, Yekbun Tutşi.
J Anim Sci Adv 2013, 3(6): 314-320.
DOI: 10.5455/jasa.20130702095554.
Online version is available on: www.grjournals.com
ISSN: 2251-7219
ARSERIM ET AL.
Original Article
Isolation, Histopathological and molecular
detection of Yesinia Pseudotuberclosis infection in
Sheep in Turkey.

1
Neval Berrin Arserim, 2sevgi Kalkanli, 3hasan İçen, 4nurettin Işık, 4yekbun Tutşi.
Dicle University Veterinary Faculty Department of Microbiology Diyarbakir/Turkey 21180.
Dicle University Medical Faculty Department of Medical Biology and Genetic .Diyarbakir /Turkey 21180.
3
Dicle University Veterinary Faculty Department of Internal Medicine Diyarbakir/Turkey 21180.
4
Laboratuary of Research, Diagnosis and Control of Animal Diseases, Microbiology, TR-21010 Diyarbakır .
5
Laboratuary of Research, Diagnosis and Control of Animal Diseases, Microbiology, TR-21010 Diyarbakır .
2
Abstract
The aim of the present study was to detect causes of abortion in sheep and early neonatal death of lambs
and treatment trial. Diagnososis of Yersinia pseudotuberculosis was made on the basis of bacteriological
from the stomach contents, lung, liver, and peritoneal fluid and pathological findings. Additionally, in order
to confirm the presence of Yersinia pseudotuberculosis nested PCR was performed. In treatment trials
Amoxycillin were given1ml/10kg BW. This is the first case report of Yersinia pseudotuberculosis causing
abortion in sheep in Turkey. As a result of this study it is suggested that Yersinia pseudotuberculosis should
be considered in the differential diagnosis of causes of ovine abortion and early neonatal death of lambs in
this country.
Key words: Yersinia pseudotuberculosis, lambs, PCR, treatment, Diyarbakir.
*
Corresponding author: Dicle University Veterinary Faculty Department of Microbiology Diyarbakir/Turkey 21180.
Received on: 19 Apr 2013.
Revised on: 21 Jun 2013.
Accepted on: 29 Jun 2013.
Online Published on: Jun 2013.
314
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
ISOLATION, HISTOPATHOLOGICAL AND MOLECULAR DETECTION…
Introduction
Yersinia pseudotuberculosis is a Gramnegative organism that causes enteritis and
septicaemia (Taffs and Glynis, 1983;
Brown and Davis 1989). Yersiniosis has
been reported from wild animals and birds,
rodents, ruminants, carnivores, non-human
primates, man and occasionally farm
animals (Brown et al., 2007; Busato et al.,
1999; Juste et al., 2009). The clinical
symptoms and pathologic changes vary
among the different host species (Zhang et
al., 2008). The common symptoms of the
disease in ruminants, and other mammals
are necrotizing, ulcerative enteritis and
mesenteric lymphadenitis with fever,
anorexia, vomiting, and diarrhea (Callinan
et al., 1988; Gill, 1966; Hum et al., 1997;
Foster et al., 2008). Septicemia due to
yersiniosis can lead to the involvement of
visceral organs, especially liver and spleen.
The infection
can lead to abortion,
stillbirth or birth of weak or healthy
newborn (Corbel et al., 1992) in sheep,
goats, pigs and cows (Novoslavskij et al.,
2010; Otter, 1996; Riet-Correa et al., 1990;
Seimiya et al., 2005; Slee and Button,
1990). Experimental infection produced a
purulent placentitis in most ewes and focal
hepatic and myocardial necrosis in
newborn lambs. Rarely, the organism
would cause other pathological changes,
such as pneumonia in cattle (Juste et al.,
2009), epididymo-orchitis of rams (Slee
and Button, 1990) or oculoglandular
syndrome in (goats Wessels et al., 2009).
The organism can be shed by
asymptomatic animals (Brown et al.,
2007).
The aim of the present study was to
detect causes of abortion in sheep and
early neonatal death of lambs.
Material and Method
Material
The materials of the study were consisted
of three fetus and four lambs which were
died approximately two days after birth
315
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
from a two hundred sheep flock in
Diyarbakir between August and November
2010. Fetuses and lambs were examined
according to standard necropsy procedures.
Tissues were collected for microbiological
examination under precautions to prevent
extraneo us or cross-contamination of
individual tissues.
Microbiology
Liver, lung and gastric contents of
aborted fetus and died lambs were cultured
on blood agar and MacConkey agar.
Culture plates were incubated at 37°C in
both aerobic and anaerobic atmospheres
for 24-48 h. Suspect Yersinia sp. colonies
were identified by the following: Gram
reaction, motility at 22 and 37°C,
fermentation of glucose, lactose. VITEK 2
GN (colorimetric) card (Biomerieux SA
France) identification kit was used for
identification.
Pathology
Three fetus and four lambs were
necropsied. Samples of liver, mesenteric
lymph node and small and large intestines
were fixed in 10% neutral buffered
formalin.
PCR assay for detection of Yersinia
pseudotuberculosis
DNA for Polimeraze Chain Reaction
(PCR) was isolated by a Purification Kit
(Vivantis, GF-BA). PCRs performed in
50μl volumes containing 5μl of template
DNA, 0,1mM each of the four
deoxynucleoside triphoshates, 5μl of 10 X
PCR buffer, 3mM MgCl2, 0,1μl of each
primer, and 0.5U of Tag DNA polymerase.
The PCR amplifications carried out at
94ºC for 5 min as an initial denaturation
step and then subjected to 30 cycles
consisting of 1 min at 94ºC,1 min at 55ºC
for detection of inv (inv-1-5'-TAA GGG
TAC TAT CGC GGC GGA-3', inv-2-5'CGT GAA ATT AAC CGT CAC ACT-3')
at 55 ºC for detection of Ypf-20210-wzz5'-GGT GAT GAG CAA GTT CAA G-3',
Ypr-20538-wzz-5'-GCT AAA TCC ACT
GCT CGC TG-3' (Bogdanovich et al.,
ARSERIM ET AL.
2003) 1 min at 72 ºC, followed by a final 5
min extension step at 72 ºC. 10μl volume
of each PCR products was loaded onto a
1.5% agarose gel containing 0.05μl/ml of
ethidium bromide solution. The gel was
visualized on an UV board. A negative
control with sterile water instead of DNA
template was also prepared. 100bp DNA
Ladder (Fermentas SM 0241) was used as
the molecular size marker.
Results
Microbiology
Yersinia pseudotuberculosis was isolated
as pure growth from the stomach contents,
lung, liver, and peritoneal fluid. Any other
bacteria or Chlamydia weren’t isolated on
tissue culture of lung, liver, kidney, brain,
and stomach contents. The caused agent
was
identified
as
Yersinia
pseudotuberculosis strains by the VITEK 2
Compact device (Fig.1). The testing of
Vitek 2 demonstrated that Yersinia
pseudotuberculosis was susceptible to
Amoxycillin, Ampicillin, Tetracyclin,
Lincocin-spectinomycin,
Sulphamethoxazole-trimetophrim
but
resistant to Eritromycin, Penicillin G.
thickness of the wall of the bowel and
abomasum, and a fibrinous or hemorrhagic
enteritis of the small and large intestine,
which was severe in the ileum.
Histopathological examinations of liver,
kidney and lung indicated to non
significant pathology. In the small and
large intestines there were multi-focal,
heavy inflammatory cell infiltrates,
consisting of neutrophils, macrophages,
lymphocytes and plasma cells within the
lamina propria.
PCR assay
Yersinia
pseudotuberculosis
was
detected with nested PCR method.
Amplified products were shown figure 2.
Figure 2. M:100 bp DNA Ladder; Lane 1,
Negative control (sterile water); Lane 2, 295
bp amplified product (inv); Lane 3, Negative
control (sterile water); Lane 4, 418 bp
amplified
product
(Ypf-20210-wzz,Ypr20538-wzz).
Figure 1. Yersinia pseudotuberculosis.
Treatment
In treatment trials bacterium was
sensitive at Amoxycillin 1ml/10kg BW
(Amoxycillin, Clamoxyl LA Pfizer).
Pathology
Three fetus and four lambs which were
necropsied, had gross lesions characterized
by peritoneal and sometimes pericardial
serous or sanguineous fibrinous fluid,
enlarged mesenteric lymph nodes, marked
edema of the mesentery, edema and
316
Discussion
In ruminants, clinical diseases by Y.
pseudotuberculosis
associated
with
abortion (Hannam, 1993; Otter, 1996),
(mastitis Juste et al., 2009) and
enterocolitis (Seimiya et al., 2005; Slee
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
ISOLATION, HISTOPATHOLOGICAL AND MOLECULAR DETECTION…
and Button, 1990). There were few reports
of Y. pseudotuberculosis abortion in sheep
UK (Baird et al., 1997), Australia
(Callinan et al., 1988; Slee and Skilbeck,
1992), New Zealand (Hodges et al., 1984)
and USA (Brown and Davis, 1989).
This study considered to be the first case
of abortion and early neonatal death of
lambs due to intrauterine Yersinia
Pseudotuberculosis infection in Turkey.
The course of the infection and its
histopathology
suggested
that
the
organisms produced a relatively slowly
evolving infection which reached the stage
of producing placental failure and fetal
death. These results indicated that strains
of Y. Pseudotuberculosis with natural
pathogenicity could produce placental
infection and abortion in pregnant sheep.
This is consistent with observations by
Karbe and Erikson, (1984), (Corbel et al.,
1992), Jerrett and (Slee, 1989). The history
of pathological signs, results of bacterial
culture and PCR of Y pseudotuberculosis
as the etiologic agent in this research
provide that there was similarities between
our results and previously reports by Gill
(1966), (Martins et al., 1998), (Iwata et al.,
2008), (Thoerner et al., 2003), Welsh and
Stair, (1993).
The epidemiology of Yersiniosis is
complex and the factors leading to clinical
disease are unknown. The organism may
be shed in the faeces by clinically normal
animals in a herd and by other species such
as rodents and birds in the environment
(Witte et al., 1985; Riet-Correa et al.,
1990). Yersinia pseudotuberculosis and
Yersinia enterocolitica have been isolated
from ovine abortion cases (Karbe and
Erikson, 1984; Otter, 1996). Infection of
ewes by Y. pseudotuberculosis can lead to
abortion, stillbirth or birth of weak or
healthy
lambs.
Infection
by
Y.
enterocolitica resulted in placentitis and
abortion,
in
subsequent
normal
pregnancies, Y. pseudotuberculosis causes
septicaemia, tissue micro-abscesses and
enteritis in lambs and young pigs and
sporadic abortions in sheep, and cattle
317
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
(Givens and Marley, 2008). At necropsy,
typical gross lesions included peritoneal
serous or sanguineous, fibrinous exudate,
enlarged mesenteric lymph nodes, marked
mesenteric oedema and fibrinous or
haemorrhagic enteritis. Histologically,
there was severe fibrinonecrotic enteritis
with mixed inflammatory cell infiltration
of
the
lamina
propria.
Y
pseudotuberculosis was isolated from the
mesenteric lymph nodes. The syndromes
occurred in winter and stress appeared to
be a contributing factor. The gross and
histopathological findings were similar to
those previously reported (Karbe and
Erikson, 1984; Otter and Callaghan, 2008)
and may assist in diagnosis.
The prevalence of Yersiniosis in
domestic animals may be higher than
reported as a consequence of the
nonspecific signs of infections as well as
the serologic cross reactions that lead to
confusion with other bacterial infections
(Hodges et al., 1984; Witte et al., 1985;
Corbel et al., 1992; Juste et al., 2009) Early
diagnosis is important for successful
treatment and reducing stress factors may
prevent clinical Yersiniosis. Tetracyclines,
neomycin, and lincomycin-spectinomycin
are reported to be effective against most
isolates of Y pseudotuberculosis and can be
used until results of antibiotic sensitivity
tests are available (Taffs and Glynis, 1983;
Bin-Kun et al., 1994). Since yersiniosis is a
potential zoonosis known to cause
gastrointestinal infections in humans, care
should be taken when handling infected
animals (Martins et al., 1998). In this study
treatment trials showed recovery in
animals were by giving antibiotics to
which the bacterium was sensitive and so
there were no more abort after treatment.
This is consistent with observations from
field researches (Sanford 1995; Juste et al.,
2009).
The epidemiology of Yersiniosis is
complex and the factors leading to clinical
disease are unknown. The organism may
be shed in the faeces by clinically normal
animals in a herd and by other species such
ARSERIM ET AL.
as rodents and birds in the environment
(Witte et al., 1985; Riet-Correa et al.,
1990). Yersinia pseudotuberculosis and
Yersinia enterocolitica have been isolated
from ovine abortion cases (Karbe and
Erikson, 1984; Otter, 1996). Infection of
ewes by Y. pseudotuberculosis can lead to
abortion, stillbirth or birth of weak or
healthy
lambs.
Infection
by
Y.
enterocolitica resulted in placentitis and
abortion,
in
subsequent
normal
pregnancies, Y. pseudotuberculosis causes
septicaemia, tissue micro-abscesses and
enteritis in lambs and young pigs and
sporadic abortions in sheep, and cattle
(Givens and Marley, 2008). At necropsy,
typical gross lesions included peritoneal
serous or sanguineous, fibrinous exudate,
enlarged mesenteric lymph nodes, marked
mesenteric oedema and fibrinous or
haemorrhagic enteritis. Histologically,
there was severe fibrinonecrotic enteritis
with mixed inflammatory cell infiltration
of
the
lamina
propria.
Y
pseudotuberculosis was isolated from the
mesenteric lymph nodes. The syndromes
occurred in winter and stress appeared to
be a contributing factor. The gross and
histopathological findings were similar to
those previously reported (Karbe and
Erikson, 1984; Otter and Callaghan, 2008)
and may assist in diagnosis.
The epidemiology of Yersiniosis is
complex and the factors leading to clinical
disease are unknown. The organism may
be shed in the faeces by clinically normal
animals in a herd and by other species such
as rodents and birds in the environment
(Witte et al., 1985; Riet-Correa et al.,
1990). Yersinia pseudotuberculosis and
Yersinia enterocolitica have been isolated
from ovine abortion cases (Karbe and
Erikson, 1984; Otter, 1996). Infection of
ewes by Y. pseudotuberculosis can lead to
abortion, stillbirth or birth of weak or
healthy
lambs.
Infection
by
Y.
enterocolitica resulted in placentitis and
abortion,
in
subsequent
normal
pregnancies, Y. pseudotuberculosis causes
septicaemia, tissue micro-abscesses and
318
enteritis in lambs and young pigs and
sporadic abortions in sheep, and cattle
(Givens and Marley, 2008). At necropsy,
typical gross lesions included peritoneal
serous or sanguineous, fibrinous exudate,
enlarged mesenteric lymph nodes, marked
mesenteric oedema and fibrinous or
haemorrhagic enteritis. Histologically,
there was severe fibrinonecrotic enteritis
with mixed inflammatory cell infiltration
of
the
lamina
propria.
Y
pseudotuberculosis was isolated from the
mesenteric lymph nodes. The syndromes
occurred in winter and stress appeared to
be a contributing factor. The gross and
histopathological findings were similar to
those previously reported (Karbe and
Erikson, 1984; Otter and Callaghan, 2008)
and may assist in diagnosis.
The prevalence of Yersiniosis in
domestic animals may be higher than
reported as a consequence of the
nonspecific signs of infections as well as
the serologic cross reactions that lead to
confusion with other bacterial infections
(Hodges et al., 1984; Witte et al., 1985;
Corbel et al., 1992; Juste et al., 2009) Early
diagnosis is important for successful
treatment and reducing stress factors may
prevent clinical Yersiniosis. Tetracyclines,
neomycin, and lincomycin-spectinomycin
are reported to be effective against most
isolates of Y pseudotuberculosis and can be
used until results of antibiotic sensitivity
tests are available (Taffs and Glynis, 1983;
Bin-Kun et al., 1994). Since yersiniosis is a
potential zoonosis known to cause
gastrointestinal infections in humans, care
should be taken when handling infected
animals (Martins et al., 1998). In this study
treatment trials showed recovery in
animals were by giving antibiotics to
which the bacterium was sensitive and so
there were no more abort after treatment.
This is consistent with observations from
field researches (Sanford 1995; Juste et al.,
2009).
This
investigation
indicates
that
Yersiniosis could be important in sheep
and lambs, especially under stressful
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
ISOLATION, HISTOPATHOLOGICAL AND MOLECULAR DETECTION…
conditions in Turkey. Further studies
should be involved more animals in
Turkey to determine the importance and
extent of Y. pseudotuberculosis infection
in lambs and sheep as well as its
prevalence and economic significance.
References
Baird, G; Caldow, G and Howie, (F 1997). Yersinia
pseudotuberculosis infection in young grazing
sheep. Proc. Sheep Vet. Soc., 21: 95-97.
Bin-Kun, H; De-Sheng, X; Hong-Bi, O; Zhang, SX
and Slee, KJ (1994). Yersiniosis in sheep due to
Yersinia enterocolitica. Br. Vet. J., 150 (5): 473479.
Bogdanovich, T; Carniel, E; Fukushima, H and
Skurnik, M. (2003). Use of O-antigen gene
cluster-specific PCR s for the identification and
O-genotyping of Yersinia pseudotuberculosis and
Yersiniapestis. J. Clin. Microbiol., 41 (11): 51035112.
Brown, CC and Davis, FN (1989). Yersinia
pseudotuberculosis enteritis in four calves. J.
Comparat. Pathol., 101: 463-466.
Brown, CC; Baker, DC and Baker, IK (2007).
Alimentary System.. In: Jubb, Kennedy and
Palmer’s Pathology of Domestic Animals. Maxie,
M.G. (Ed.). 5th Edn., Saunders Ltd. USA. pp:
132-206.
Busato, A; Hofer, D; Lentze, T; Gaillard, C and
Burnens, A (1999). Prevalence and infection risks
of zoonotic enteropathogenic bacteria in Swiss
cow-calf farms. Vet. Microbiol., 69: 251-263.
Callinan, RB; Cook, RW; Boulton, JG; Fraser, GC
and Unger, DB (1988). Enterocolitis in cattle
associated with Yersinia pseudotuberculosis
infection. Australian. Vet. J., 65: 8-11.
Corbel, MJ; Ellis, B; Richardson, C and Bradley, R
(1992). Experimental Yersinia enterocolitica
placentitis in sheep. Br. Vet. J., 148:339-349.
Foster, AP; Otter A and Bolger, (VA 2008).
Yersinia pseudotuberculosis infection in a suckler
calf in the United Kingdom: A case report. Vet.
J., 175(1):139-140.
Gill, J; (1966). Yersiniosis of farm animals in New
Zealand. Surveillance., 23 (4): 24-26.
Givens, MD; and Marley, (MS 2008).
Infectious causes of embryonic and fetal
mortality. Theriogenology., 70 (3): 270-85.
Hannam, DA (1993). Bovine abortion associated
with Yersinia pseudotuberculosis. Vet. Rec., 133:
372.
Hodges, RT; Carman, MG and Mortimer, WJ
(1984). Serotypes of Yersinia pseudotuberculosis
recovered from domestic livestock. New Zealand.
Vet. J., 32: 11-13.
319
J. Anim. Sci. Adv., 2013, 3(6): 314-320.
Hum, S; Slattery, S and Love, SC (1997). Enteritis
associated with Yersinia pseudotuberculosis
infection in a buffalo. Aust. Vet. J., 75 (2): 95-97.
Iwata, T; Une, Y; Okatani, AT; Kato, Y and
Nakadai IA (2008). Virulence characteristics of
Yersinia
pseudotuberculosis
isolated from breeding monkeys in Japan. Vet.
Microbiol., 129: 404-409.
Jerrett,
IV
and
Slee,
(KJ
1989).
Bovine abortion associated with Yersinia
pseudotuberculosis infection. Vet. Pathol., 26
(2):181-183
Juste, RA; Minguijona, E; Arranz, J; Fuertes, M
and Beltran de Heredia, I (2009). Lamb mortality
in an outbreak of Yersinia pseudotuberculosis
mastitis, as a collateral effect of colostrum
feeding for Lentivirus-control. Small. Ruminant
Res., 86: 46-51
Karbe, E; and Erikson, (ED 1984). Ovine abortion
and stillbirth due to purulent placentitis caused by
Yersinia pseudotuberculosis. Vet. Pathol., 21:
601-606.
Martins, CHG; Bauab, TM and Falcao, (DP 1998).
Characteristics of Yersinia pseudotuberculosis
isolated from animals in Brazil. J. Applied
Microbiol., 85: 703-707.
Novoslavskij, A; Kabasinskiene, A; Korkeala, H
and Malakauskas, M (2010). Prevalence of
Yersinia
enterocolitica
and
Yersinia
pseudotuberculosis in slaughtered pigs within 5
months period in lithuania veterinarija ir
zootechnika. Vet. Med. Zoot. T., 51 (73): 30-35.
Otter, A (1996). Ovine abortion caused by Yersinia
pseudotuberculosis. Vet. Rec., 138: 143-144
Otter, A and Callaghan, G (2008). Yersiniosis in
lambs on fodder beet. Vet. Rec., 162(21):699
Riet-Correa, F; Gil-Turnes, C; Reyes, JC; Schild,
AL and Mendez, MC (1990). Yersinia
pseudotuberculosis infection of buffaloes (Bubalu
s bubalis). J. Vet. Diagn. Invest., 2 (1): 78-9
Sanford,SE
(1995).
Outbreaks of yersiniosis causedby Yersinia
pseudotuberculosis in farmed cervids. J. Vet.
Diagn. Invest., 7 (1): 78-81.
Seimiya, YM; Sasaki, K; Satoh, C; Takahashi, M;
Yeagashi, G and Iwane, H (2005). Caprine
enteritis
associated
with
Yersinia
pseudotuberculosis infection. J. Vet. Med. Sci.,
67: 887-890.
Slee, KJ and Button, C (1990). Enteritis in sheep,
goats and pigs due to Yersinia pseudotuberculosis
infection. Australian Vet. J., 67: 320-322.
Slee, KJ and Skilbeck, NW (1992). Epidemiology
of Yersinia pseudotuberculosis and Y.
enterocolitica infections in sheep in Australia. J.
Clin. Microbiol., 30: 712-715.
Taffs, LF and Glynis, D (1983). An outbreak of
Yersinia pseudotuberculosis infection in a small
indoor breeding colony of red-bellied (Saguinus
labiatus) tamarins. Lab. Anim., 17: 311-320
ARSERIM ET AL.
Thoerner, P; Bin Kingombe, CI; Bogli-Stuber, K;
Bissig-Choisat, B; Wassenaar, TM; Frey, J and
Jemmi, T(2003). PCR detection of virulence
genes
in
Yersinia
enterocolitica
and Yersinia pseudotuberculosis
and
investigation of virulence gene distribution.
Applied Environ Microbiol., 69 (3):1810-1816.
Zhang, S; Zhang, Z; Liu, S; Bingham, W and
Wilson,
F
(2008).
Fatal yersiniosis in farmed deer caused
by Yersinia pseudotuberculosis serotype O:3
Encoding a mannosyltransferase-like protein
WbyK. J. Vet. Diagn. Invest., 20 (3):356-359.
Welsh, RD and Stair, EL (1993). Yersinia
pseudotuberculosis bovine abortion. J. Vet.
Diagn. Invest, 5: 109-111.
Wessels, ME; Payne, JH and Bannerman, RP
(2009). Oculoglandular syndrome caused by
Yersinia pseudotuberculosis in a dairy goat. J.
Comp. Pathol., 141: 190-194.
Witte, ST; Sponenberg, DP and Collins, TC (1985).
Abortion and early neonatal death of kids
attributed
to
intrauterine
Yersinia
pseudotuberculosis infection. JAVMA 15;
187(8):834-834.
320
J. Anim. Sci. Adv., 2013, 3(6): 314-320.