Shepherd dogs as a common source for Salmonella enterica serovar Reading in Garmsar, Iran

102
http://journals.tubitak.gov.tr/veterinary/
Turkish Journal of Veterinary and Animal Sciences Turk J Vet Anim Sci
(2013) 37: 102-105
© TÜBİTAK
doi:10.3906/vet-1107-1
Shepherd dogs as a common source for Salmonella enterica serovar
Reading in Garmsar, Iran
Taghi ZAHRAEI SALEHI1, Mahdi ASKARI BADOUEI2,*, Omid MADADGAR1, Seyed Reza GHIASI3, Iradj ASHRAFI TAMAI1
1
Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2
Department of Pathobiology, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran
3
Department of Clinical Science, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran
* Correspondence: askarim@vetmed.ut.ac.ir
Salmonellosis is one of the most important zoonotic
diseases with global distribution and importance. Infection
can occur through the consumption of contaminated
foodstuffs or contact with infected animals (1–3). Carrier
states in animals are of major concern in the epidemiology
of salmonellosis, because animals can become the latent
carriers of Salmonella serovars and shed the organism
into the environment without any apparent clinical signs.
Although Salmonella can cause enteritis and diarrhea in
dogs, most infected animals show no sign of disease in
spite of infection and fecal shedding (4,5). Salmonella
serovars readily colonize in the canine large intestine and
mesenteric lymph nodes. Fecal shedding of the pathogen
in naturally occurring infections probably continues for a
period of at least 6 weeks. Since the lymph nodes harbor
the agent, the carrier state may persist for much longer
periods (6).
Among different species, dogs can be one of the most
important reservoirs of Salmonella for the following
reasons. Dogs are one of the most popular pets in close
contact with their owners, including children, and they
can shed the organism for weeks without any clinical signs.
They are also the inseparable companion of food animals
in the nonintensive sheep raising system, which is still
common in many countries.
The aim of the present study was to investigate the
role of herd dogs in the epidemiology of salmonellosis.
Although Salmonella infection has been investigated
previously in dogs, this appears to be the first study that
primarily assesses the potential role of shepherd dogs in
the epidemiology of salmonellosis.
Fecal samples were taken from 38 clinically healthy
mixed-breed shepherd dogs related to 19 sheep flocks in
Garmsar Province, which is located 90 km to the east of
Tehran, the capital city of Iran. Fecal samples were taken
using sterile swabs and sent to the laboratory within 5
h. The samples were subsequently cultured in 10 mL of
selenite F broth (Merck, Germany) and incubated for 12 to
16 h at 37 °C. Following enrichment, a loopful of selenite
broth culture was streaked onto selective media, including
xylose lysine deoxycholate agar (Merck) and MacConkey
agar (Merck). After incubation, the plates were examined
and 3 suspected colonies were picked and subcultured, and
the isolates were finally tested by means of conventional
biochemical tests (7).
Abstract: Salmonella infection is one of the most important diseases that affect all animal species and is the leading cause of foodborne
infections worldwide. One of the challenges in the control and prevention of salmonellosis is the recognition of potential carriers. To
assess the potential role of shepherd dogs in the epidemiology of salmonellosis, fecal samples were taken from 38 shepherd dogs in
Garmsar Province. Salmonella strains were isolated from 4 dogs (10.5%) related to 3 sheep flocks. All of the strains were confirmed
at the genus level using invA virulence gene PCR. Serotyping confirmed all of the strains as Salmonella Reading. To study the clonal
relatedness of the isolates, the strains were subjected to RAPD-PCR, and antimicrobial resistance patterns were also determined using
9 antibacterials. The similar RAPD-PCR profiles and resistance patterns strongly suggested that the shepherd dogs in the present study
shed a similar clone of S. Reading; therefore, shepherd dogs can be recognized as a common source for this serotype in Garmsar.
Key words: Salmonellosis, shepherd dogs, Garmsar, Salmonella Reading, RAPD-PCR
Received: 01.07.2011 Accepted: 22.02.2012 Published Online: 22.01.2013 Printed: 22.02.2013
Short Communication
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ZAHRAEI SALEHI et al. / Turk J Vet Anim Sci
The biochemically identified strains were subjected to
polymerase chain reaction (PCR) for theinvAgene, using the
S139 (5’-GTGAAATTATCGCCACTGTCGGGCAA-3’)
and S141 (5’-TCATCGCACCGTCAAAGGAACC-3’)
primers to confirm the isolates at the genus level (3).
Reactions with these primers were carried out in a 25-µL
amplification mixture consisting of 2.5 µL of 10X PCR
buffer (500 mM KCl, 200 mM Tris–HCl), 0.5 µL of dNTP
mix (10 mM), 2 mM MgCl2, 0.5 µM of each primer, 1 U of
Taq DNA polymerase, and ultrapure water.
Serotyping of the isolated bacteria, which were
confirmed at the genus level by biochemical tests and the
presence of the invA virulence marker, was performed
using commercial antisera (Difco, USA) according to the
manufacturer’s instructions.
Resistance profiles of the isolated Salmonella strains
were determined on Mueller-Hinton agar (Merck) using
the Kirby–Bauer method according to Clinical and
Laboratory Standards Institute protocols (formerly the
National Committee for Clinical Laboratory Standards).
Nine antibacterial disks were used, including ampicillin
(AM; 10 µg), penicillin G (P; 10 units), streptomycin
(S; 10 µg), erythromycin (E; 15 µg), Linco-Spectin (LP;
150 µg), chloramphenicol (C; 30 µg), trimethoprimsulfamethoxazole (SXT; 1.25/23.75), ceftriaxone (CRO; 30
µg), and cefixime (CM; 5 µg).
To assess the clonal relatedness of the isolates,
random amplification of polymorphic DNA (RAPD)-
PCR profiles were investigated using 3 sets of arbitrary
primers, including P1254 (5’-CCGCAGCCAA-3’),
23L (5’-CCGAAGCTGC-3’), and OPA-4
(5’-AATCGGGCTG-3’) (8,9). In brief, a single colony of
each isolate from an agar plate was picked and suspended
in 200 µL of distilled water. After vortexing, the suspension
was boiled for 5 min, and 50 µL of the supernatant was
collected after centrifuging for 10 min at 15,000 × g. The
DNA concentration of the boiled extracts was determined
with a spectrophotometer and adjusted to approximately
40 ng DNA/µL. The PCR was conducted in a 25-µL
volume containing 40 ng of template DNA, 2 mM MgCl2,
0.5 µM of primer, 1 U of Taq DNA polymerase (Fermentas,
Lithuania), and 200 µM of each dNTP in 1Χ PCR buffer.
A Salmonella Typhimurium strain (Microbial Collection,
Faculty of Veterinary Medicine, Tehran University)
was used as the positive control and distilled water was
used as the negative control. The PCR products were
electrophoresed on 2% agarose gel at 80 V for 2 h and
visualized after staining with ethidium bromide.
Salmonella spp. was isolated from 4 shepherd dogs
(10.5%) belonging to 3 sheep flocks. In other words,
15.8% of sheep flocks in the area have carrier shepherd
dogs. All 4 of the isolates produced the 284-bp specific
product of the invA gene. Serotyping of the strains using
commercially available antisera identified all isolates
to be S. enterica subsp. enterica serovar Reading. Four
strains revealed similar resistance patterns; in fact, all of
the strains were resistant to streptomycin, trimethoprimsulfamethoxazole, penicillin, and erythromycin, but
sensitive against other applied antimicrobials, including
ampicillin, Linco-Spectin, chloramphenicol, ceftriaxone,
and cefixime. The molecular typing by the RAPD method
using 3 sets of primers could not discriminate the isolate
genetically and a similar RAPD-type was observed in 4
isolated Salmonella strains (Figures 1–3).
Traditional nonintensive sheep raising systems rely on
the presence of shepherd dogs as inseparable members of
the flock. This traditional system is still fairly common in
Middle Eastern countries, and also in some parts of Europe
and Australia. Shepherd dogs are usually born near their
flocks and live with ruminants through their working life.
Therefore, direct contact of these dogs with food animals
is unavoidable. Dogs are among the most famous latent
carriers of Salmonella serovars because they can shed the
organism for 3 to 4 weeks, and in rare cases for up to 100
days (10,11). On the other hand, a single sheep flock can
provide meat and dairy products for hundreds of people
or consumers, and this is when the importance of a single
infected dog in a flock seems to be overlooked. Results of
this study indicated that shepherd dogs can be a potential
reservoir for Salmonella spp. and the transmission of the
disease to food animals. Although disease transmission
Figure 1. RAPD-PCR (Primer 1254): M, Marker, 100 bp-plus;
C, S. Typhimurium; Lanes 1–4, S. Reading isolated from 4 herd
dogs in this study.
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ZAHRAEI SALEHI et al. / Turk J Vet Anim Sci
can occur as a blind circle from sheep and goat to dogs, and
from dogs to the animals of the flock, this epidemiological
circle may occur several times in a single flock over a long
period of time. Furthermore, the feeding of herd dogs with
raw meat in urban areas is fairly common; this can lead
to higher rates of infection and shedding of Salmonella
serovars. Finley et al. (4) showed that feeding dogs raw
foods that are contaminated by Salmonella spp. can be a
major public health threat to humans and animals. Poorquality foods have also been considered as an important
source of salmonellosis in dogs (12). Previously, the
Salmonella spp. shedding of dogs of the same geographical
area (Garmsar Province) not related to herds was studied,
but Salmonella spp. was not isolated (unpublished study).
As mentioned earlier, this may be due to the eating of raw
foods and also to intimate contact with ruminants.
The present study indicated the presence of Salmonella
spp. in 10.5% of shepherd dogs in Garmsar Province. The
shedding pattern of Salmonella serovars in animals is
usually intermittent and periodical; therefore, 3 rounds of
sampling are recommended to assess the carrier states in
animals. In this study, taking only one sample per animal
was possible, and as a result of that, the shedding status
rate in the herd dogs may be underestimated. To our
knowledge, there is no report or study available on the
prevalence of salmonellosis in herd dogs, but interestingly,
S. Reading has been frequently reported in both small
ruminants and dogs. In Italy, S. Reading was one of the
most frequent Salmonella serovars isolated from dogs
(13). In another study in Ethiopia, S. Reading was one of
the most prevalent serovars isolated from sheep and goats
(14). In 1991, the Centers for Disease Control reported a S.
Reading-associated outbreak of salmonellosis in humans;
the outbreak was reported to be caused by the consumption
of improperly cooked turkey meat (15). White et al. (16)
presented a study on dog treats derived from samples
such as pig ears and other animal parts and followed up
on isolated Salmonella spp. for their epidemiologic sources
using serotyping, pulsed-field gel electrophoresis, and
antimicrobial susceptibility testing. Their results indicated
that animal-derived dog treats in the United States could
be a potential source of animal and human infections with
Salmonella spp., including multidrug-resistant Salmonella
strains.
A noteworthy observation in the present study
was the shedding of clonally similar S. enterica subsp.
enterica serovar Reading in herd dogs. This finding
suggested that the herd dogs act as a common source
for a particular serotype of Salmonella in the studied
area. There is no authentic information available on the
epidemiology of salmonellosis in humans and animals in
Garmsar Province. Further study on the prevalence and
epidemiology of salmonellosis in Garmsar Province can
lead to deeper insight into the importance of herd dogs in
the transmission and epidemiology of salmonellosis.
Figure 2. RAPD-PCR (Primer OPA4): M, Marker, 100 bp-plus;
C, S. Typhimurium; Lanes 1–4, S. Reading isolated from 4 herd
dogs in this study.
Figure 3. RAPD-PCR (Primer 23L): M, Marker, 100 bp-plus; C,
S. Typhimurium; Lanes 1–4, S. Reading isolated from 4 herd dogs
in this study; C–, Negative control.
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ZAHRAEI SALEHI et al. / Turk J Vet Anim Sci
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