Equine salmonellosis in southern Brazil

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REGULAR ARTICLES
Equine salmonellosis in southern Brazil
Gregory Duarte Juffo1 & Daniele Mariath Bassuino1 & Danilo Carloto Gomes2 &
Fabiana Wurster1 & Caroline Pissetti3 & Saulo Petinatti Pavarini1 & David Driemeier1
Received: 15 February 2016 /Accepted: 16 December 2016 /Published online: 24 December 2016
# Springer Science+Business Media Dordrecht 2016
Abstract The Salmonella sp. genus is identified in several
species, and the zoonosis it causes is one of the most im-
portant types worldwide. The specifics of salmonellosis vary
according to the function of the serovar involved, the species
affected, age and predisposing factors. However, few cases
of equine salmonellosis have been reported. This study pre-
sents ten confirmed salmonellosis cases in equines in south-
ern Brazil. Six were adult animals with stress factors preced-
ing the disease, while four were foals, three of which pre-
sented with hyperacute manifestations. The main clinical
signs were diarrhea, anorexia, and hyperthermia. Lesions
varied in distribution and severity, although fibrinonecrotic
or necrohemorrhagic enteritis was observed in all animals,
mainly in the large intestine (large colon and cecum—8/10)
and small intestine (3/10). Substantial liquid content, mainly
hemorrhagic, was observed in all animals. The most charac-
teristic microscopic lesion was mucosa necrosis, which is
often accompanied by fibrin deposition, followed by necro-
sis of follicular centers and vascular changes. Bacterial iso-
lation revealed seven isolates. Five were serotyped, and the
serovars Typhimurium and Anatum were associated with
two cases each, while Muenster was associated with a case
whose lesion pattern varied. Immunohistochemical staining
was positive in all cases. All diagnoses were based on the
clinical history, macroscopic and histological lesions, and
the bacterial isolation and/or immunostaining associated
with histological lesions.
Keywords Enteritis . Immunohistochemistry . Infectious
diseases
Introduction
Equines salmonellosis is an important infectious disease
caused by different Salmonella serovars, that affects animals
of all ages (Walker et al. 1991; Chapman, 2009). In adult
equines, it occurs sporadically, although outbreaks have been
reported in animals at a higher risk of contamination or ex-
posed to stress factors (Collett and Mogg, 2004). Such cases
occur mainly in veterinary hospitals, with higher prevalence in
debilitated or sick animals that also present acute diarrhea
(Radke et al. 2002; Schauser et al. 2004). In Brazil, it is esti-
mated that the horse population comprises five million indi-
viduals and southern Brazil accounts for 18.2% of the total
horse population (Lima and Cintra 2015).
Data on equine salmonellosis in Brazil are scarce, and it is
very likely that the disease is misdiagnosed and
underreported. This study describes clinical, pathological
and epidemiological aspects associated with naturally ac-
quired equine salmonellosis cases in southern Brazil and dis-
cusses the applicability of immunohistochemistry (IHC) to
detect the presence of Salmonella sp.
* David Driemeier
davetpat@ufrgs.br
1 Department of Clinical Pathology, College of Veterinary Medicine,
Federal University of Rio Grande do Sul (UFRGS), Avenida Bento
Gonçalves, no. 9090, 91540-000 Porto Alegre, RS, Brazil
2 Laboratory of Pathological Anatomy, College of Veterinary
Medicine and Zootecnia, Federal University of Mato Grosso do Sul
(UFMS), Campo Grande, Mato Grosso do Sul, Brazil
3 Department of Preventive Veterinary Medicine, College of
Veterinary Medicine, Federal University of Rio Grande do Sul
(UFRGS), Porto Alegre, Rio Grande do Sul, Brazil
Trop Anim Health Prod (2017) 49:475–482
DOI 10.1007/s11250-016-1216-1
Materials and methods
Equine necropsy archives from 2003 and 2012 were reviewed
of the Veterinary Pathology Service, Federal University of Rio
Grande do Sul (SPV-UFRGS). The following criteria from
Salmonella sp. infection diagnosis they were adopted: pres-
ence of intestinal lesions, positive bacterial isolation, and/or
positive IHC. Table 1 shows the age, breed, sex, and clinical
history of the selected equines.
Necropsy was carried out in all equines within 6 to 24 h
after death. Multiple samples of the organs and central ner-
vous system were collected and fixed in 10% neutral buffered
formalin. Tissues samples were trimmed, processed routinely
for histopathology, and stained with hematoxylin and eosin.
Immunohistochemistry
Selected intestine, liver, and mesenteric lymph node sec-
tions were mounted on positive slides (Easy-Path®,
Erviegas, SP, Brazil). Endogenous peroxidase blocking
was carried out by incubating slides in a 10% hydrogen
peroxide solution in methanol. Antigen recovery was
conducted with protease XIV (Sigma® Chemical
Company, Poole, UK) for 15 min. To reduce nonspecific
background binding, sections were treated with 5%
skimmed milk (Molico®) dissolved in distilled water.
Sections were then incubated with the polyclonal primary
anti-Salmonella antibody (Biogenesis®) diluted to
1:1000 in phosphate buffer saline (PBS) and treated with
secondary biotinylated antibody for 20 min and with
streptavidin-peroxidase conjugate (LSAB, Dako,
Carpinteria, California, USA®) for an additional
20 min. Next, sections were treated with chromogen
(AEC + High Sensitivity, Dako) for 2 min and then
counterstained with Mayer’s hematoxylin for less than
1 min, washed, and mounted on aqueous medium
(Schauser et al. 2004). Positive controls previously con-
firmed (Watanabe et al. 2011) were prepared simulta-
neously. Negative controls included sections of intestines
of equines that died with no enteric signs and that
Table 1 Identification, history,
and clinical signs in 10 equines
diagnosed with salmonellosis in
southern Brazil
Animal Age Breed Gender History and clinical signs
1 6 months Mixed Female Three foals, in the same farm, presented with fetid, yellowish
diarrhea, hyperthermia, prostration, and anorexia, and this
animal died within 24 h.
2 3 months Mangalarga Male Animal found dead with no clinical signs.
3 5 years Crioula Male Admission to the hospital, for 4 days, for elective
orchiectomy. After discharge, the animal presented with
anorexia, hyperthermia, reddish urine, and diarrhea for
5 days with no improvement with antibiotic treatment.
The animal was euthanized.
4 7 years Mixed Male Equines 4 and 5were from the same farm andwould take part
in a rodeo. On the day after they arrived to the venue, they
presented anorexia and apathy, which evolved to colic,
restlessness, intense sweating, and hyperthermia. The
animals were euthanized.
5 10 years Mixed Female
6 Adult Mixed Female Colic. Laparostomy. Death 6 h after surgery.
7 8 years Crioula Male Two days of anorexia, which was followed by admission to
the hospital with subsequent hyperthermia, intense
lacrimation, blood in the urine, diarrhea, and progression
to shock. The animal was euthanized.
8 7 months Crioula Female Severe respiratory failure with bilateral increase in the neck
(lymph node region) and treatment with azithromycin.
Referred to a clinic where the animal presented diarrhea
for 12 days until death.
9 3 years Mixed Male Admission to the hospital for 12 days to treat an olecranon
fracture on a left leg. After 12 days, the animal was
brought back with suspected circulation shock, abdominal
pain and diarrhea; clinical exams showed cyanotic
mucosae, leukopenia, and cold extremities. The animal
was euthanized.
10 40 days Mixed Female Found dead without clinical signs. In the same farm, there
were 2 mares, with respective foals, 1 of which presented
with signs of intestinal infection and was isolated for
treatment.
476 Trop Anim Health Prod (2017) 49:475–482
presented with enteric lesions (incubated in PBS, replac-
ing the primary antibody).Bacteriological examination
Samples (intestine, liver, and mesenteric lymph nodes) were
submitted to bacterial isolation in the Preventive Veterinary
Medicine Department, UFRGS. A fragment of each sample
was inoculated in Tryptone Soy agar (TSA) (Oxoid) supple-
mented with 5% ovine blood and incubated at 35 ± 2 °C for
24/48 h. Samples that exhibited bacterial growth were trans-
ferred to TSA agar for pure isolation and then subjected to
biochemical tests (Michael et al. 2003). In parallel, a fragment
of each sample was transferred to Muller-Kauffmann
tetrathionate broth and Rappaport-Vassiliadis broth (selective
enrichment). Aliquots of each broth were seeded in Xylose-
Lysine-Tergitol4 agar (XLT4) and Brilliant Green Phenol Red
Lactose Sucrose agar (BPLS). Characteristic colonies were
transferred to TSA agar for pure isolation and subjected to
biochemical tests (Michael et al. 2003). Isolates that exhibited
biochemical profiles compatible with Salmonella sp. were
confirmed by the agglutination test using a polyvalent serum
against Salmonella sp. (Probac, São Paulo) and sent for
serotyping in the Oswaldo Cruz Institute (FIOCRUZ).
Results
Gross lesions
Macroscopic changes were observed in the large intestines of
all equines. Lesions were diffusely hyperemic (6/10).
Additionally, differently sized, segmented, well-defined areas
were observed in the serosa extending to the mucosa, which
was dark red, brittle, and ulcerated (Fig. 1a). The mucosa was
thickened and hyperemic (5/10), wrinkled, and finely granular
(2/10). Apart from these changes, only transmural gelatinous
edema (2/10) was observed, which, in one case, was associat-
ed with multifocal ulceration areas (1/10). Yellow-brown
pseudomembranes adhering to the mucosa (3/10) (Fig. 1b)
were detected, as were button-shaped ulcers covered with a
fibrin layer that presented with a peripheral hyperemic halo
(1/10) (Fig. 1c). In all animals, green to brown-red intestinal
contents were abundant and liquid. The main lesions in the
small intestines were hyperemia, petechiae, and ecchymoses,
which were diffusely distributed in the serosa. In three ani-
mals, the main finding was observed in this intestinal section.
These lesions were characterized by brown-red liquid con-
tents, large levels of free fibrin in the intestinal lumen
(Fig. 1d), and the deposition of yellow pseudomembranes in
Fig. 1 a Abdominal cavity;
equine 6. Large colon segments
with segmented dark red serosa
areas. b Large colon and cecum;
equine 3. Ventral colon (open);
thick yellow-brown material
diffusely adhered to the mucosa. c
Large colon and cecum; equine 9.
The colon (open) shows ulcers
that are covered with fibrin in the
yellow central portion and white
peripheral portion; hyperemia
halo. d Small intestine, jejunum;
equine 4. The serosa surface is
diffusely dark; abundant, brown-
red content with substantial free
fibrin in the lumen. e Small
intestine and jejunum; equine 5.
Mucosa covered with fibrillar,
yellowish material
(pseudomembrane); intense
mucosa hyperemia. f Kidney;
equine 7. Capsular surface with
multiple red dots between 0.2 and
0.5 cm in diameter
Trop Anim Health Prod (2017) 49:475–482 477
the mucosa (2/10) (Fig. 1e). In one animal, the serosa present-
ed with dark red scattered areas between 3 and 10 cm in
length, which corresponded to the deposition of fibrin in the
mucosa and hemorrhage on Peyer’s patches. Table 2 lists the
macroscopic changes in the small and large intestines of the
ten equines. Changes in the other organs were diverse and of
different intensity, such as intense hemorrhage in the adrenal
cortex (6/10) and petechiae and ecchymoses in the kidneys
(Fig. 1e), lungs, mesentery, trachea, heart, skeletal muscle,
and subcutaneous tissue.
Microscopic lesions
Histologically, intestinal lesions were classified as necrotic,
fibrinonecrotic, or necrohemorrhagic in all equines. These
lesions were observed mainly in the large intestine (large
colon and cecum) and in the small intestine (3/10). The main
changes were surface necrosis of the mucosa, which was
sometimes transmural, apart from moderate to severe hyper-
emia, hemorrhage, and thrombosis. The inflammatory infil-
trate mainly consisted of neutrophils, but macrophages, lym-
phocytes, plasmocytes, and eosinophils were also present.
Diphtheritic membranes associated with mucosa necrosis
were observed in the large intestine (4/10) and small intes-
tine (2/10) (Fig. 2a–c). Fibrin thrombi, edema, necrosis of
lymphoid follicles, and lymph vessel dilation were observed
in the submucosa in most animals. Hyperemia of the mucosa
and submucosa (4/10) were detected, as was fibrinoid necro-
sis of blood vessels of the submucosa (2/10). The intensity
of these lesions is shown in Table 3. Histological lesions in
other organs were wide-ranging, including Bparatyphoid
nodules^ (4/10) and thrombosis (5/10) in the liver; hemor-
rhage and alveolar edema in the lungs (5/10); thrombosis
(2/10) and mineralization of the alveolar wall (1/10); severe
diffuse hemorrhage in adrenal glands (5/10) with foci of
coagulation necrosis (1/10); subepicardial and subendocardi-
al hemorrhage in the heart (3/10); cardiomyocyte necrosis,
which at times was mineralized and presented with light
mononuclear infiltrate (1/10); microthrombi and severe hem-
orrhage in the glomerulus with finely granular amorphous
eosinophilic material (protein) in the tubules and tubular
necrosis with multifocal hemorrhage (1/10); and lymphoid
tissue necrosis and multifocal infiltrate of macrophages in
mesenteric lymph nodes (2/10) and in the white pulp (1/10).
Immunohistochemistry
All animals were positive in IHC using the polyclonal pri-
mary anti-Salmonella sp. antibody. Positive staining was
observed in the large intestine of eight equines (1, 2, 3,
6, 7, 8, 9, and 10) and in the small intestine of four animals
(1, 4, 5, and 8). The intensity and distribution varied from
diffuse and accentuated (in the surface mucosa, sometimes
extending to the large intestine’s lamina propria; Fig. 2d) to
moderate in the villi of the small intestine and Peyer’s
patches. Positive staining was also observed in free macro-
phages in the lamina propria inside necrotic crypts and cell
remains (Fig. 2e, f). In two cases, a positive IHC reaction
was observed in the mesenteric lymph nodes.
Table 2 Macroscopic changes in the small and large intestines in 10 equines with salmonellosis in southern Brazil
N° Small intestine Large intestine
Serosa Mucosa Serosa Mucosa
1 N.C. N.C. N.C. Irregular surface, granular aspect
2 N.C. N.C. N.C. Irregular surface, granular aspect
3 N.C. N.C. Reddish / hyperemic with focal
segmented hemorrhage.
Thickened, hyperemic with ulcers and covered
by pseudomembrane
4 Hyperemic with petechiae
and ecchymosis
Hyperemic, thickened,
pseudomembrane.
Reddish. Edematous
5 Hyperemic with petechiae and
ecchymosis.
Hyperemia, pseudomembrane. Reddish. Edematous
6 N.C. N.C. Reddish / hyperemic, with
intense focal segmented
hemorrhage.
Thickened and hyperemic with dark red
multifocal areas covered with pseudomembrane
7 Hyperemic with petechiae
and ecchymosis.
Hyperemic with fibrin
deposition on Peyer’s
patches.
Reddish. Thickened and hyperemic
8 N.C. N.C. N.C. Edematous with ulceration areas covered with
pseudomembrane
9 N.C. N.C. Reddish / hyperemic with focal
segmented hemorrhage.
Thickened and hyperemic with button-like ulcer
multifocal areas and covered with fibrin
10 Hyperemic N.C. N.C. Hyperemic, edematous and finely granular
N.C. no change
478 Trop Anim Health Prod (2017) 49:475–482
Bacterial isolates
Bacterial examination resulted in the isolation of Salmonella
sp. in seven out of ten cases. Isolates from five cases were
submitted for further characterization at the Oswaldo Cruz
Institute. Serotyping revealed the presence ofS. anatum (2),
S. typhimurium (2), and S. muenster (1). Bacterial isolation
with the corresponding serotypes is shown in Table 4.
Discussion
Clinical signs, macroscopic and histologic changes observed
in the present study are similar to previous findings for
equine salmonellosis (Gibbons, 1980). Together with the
results of bacterial isolation (in seven cases) and/or positive
IHC staining with anti-Salmonella antibody (observed in all
cases), these clinical findings are essential in the diagnosis
of Salmonella sp. infections (Collett and Mogg, 2004; Feary
and Hassel, 2006). The severity of a Salmonella sp. infection
in equines varies with age, predisposing factors, and the
serotype involved. The most severe signs are observed in
foals younger than 4 months of age, in which septicemia
is more common than in adult equines (Radostits et al.
2007; Chapman, 2009). In the present study, 75% of the
cases in foals were the hyperacute form of the disease, with
short, severe clinical progression that ended in death due to
circulatory shock.
Fig. 2 a Large intestine (large colon); equine 3. Thick diphtheritic
membrane formed by abundant fibrin, cell debris, intense inflammatory
cell infiltrates and bacterial volumes associated with diffuse mucosa
necrosis; severe capillary thrombosis; mucosa congestion (hematoxylin-
eosin). b Small intestine; equine 4. Diphtheritic membrane formed by
abundant fibrin, cell debris, degenerated neutrophils, and bacterial
volumes associated with diffuse mucosa necrosis (hematoxylin-eosin). c
Large intestine (large colon); equine 3. Necrosis of cryptae and epithelial
desquamation; some crypts are filled with cell debris; predominantly
neutrophilic infiltrate (hematoxylin and eosin). d Large intestine (large
colon); equine 3. Immunohistochemistry with the polyclonal primary
anti-Salmonella antibody shows diffuse bacterial antigens in the surface
mucosa, sometimes as far as the lamina propria and submucosa
(streptavidin-peroxidase conjugate;chromogen AEC). e Large intestine
(large colon); equine 1. Immunohistochemistry with polyclonal primary
anti-Salmonella antibody shows bacterial antigens in the surface mucosa
(streptavidin-peroxidase conjugate; AEC chromogen). f Large intestine
(large colon); equine 1. Larger magnification, immunoreaction in free
macrophages in the lamina propria, inside necrotic cryptae and cell
debris (polyclonal primary anti-Salmonella antibody; streptavidin-
peroxidase conjugate; AEC chromogen)
Trop Anim Health Prod (2017) 49:475–482 479
Studies of salmonellosis in horses have identified risk fac-
tors that increase the likelihood of infection during hospitali-
zation (Hird et al. 1986; Kim et al. 2001; Ernst et al. 2004).
Despite some inconsistencies between these studies, possibly
because of the differences in sample populations and sampling
techniques, many similar risk factors exist. Horses that re-
ceived antibiotic therapy and were undergoing feed restriction
or diet change were at greater risk for developing salmonello-
sis. This is most likely because of alterations in enteric micro-
flora (House et al. 1999; Ernst et al. 2004). Additionally, high
bacterial charge can be released by asymptomatic carriers,
which increases the contamination of hospital environments
and consequently favors the ingestion of large doses of
Salmonella in animals especially accommodated in small
spaces. Thus, nosocomial infections in the postoperative
period may occur without any connection with pre-existing
enteric diseases (Gibbons, 1980; Brown et al. 2007;
Chapman, 2009). Of the six adult equines investigated in the
present study, four were hospitalized for surgery, while two
were transported together with other animals. We believe that
the dosage and intake of Salmonella are the main reasons why
animals succumb to the infections.
Diarrhea was the most common clinical sign in the equines
that were examined. It results from the release of bacterial
endotoxins that are mediated by hyperstimulation and by the
inflammatory response of the host. These events may in turn
cause microvascular changes in the cecum and colon, with
systemic hypoproteinemia and local edema in the mucosa
and submucosa (Oliver and Stämpfli, 2006). In the present
study, such alterations were histologically observed in all ten
cases.
Lesions observed during postmortem examination were
similar to those reported in previous studies. These lesions
were mainly typhlocolitis (Roth 1988; Astorga et al. 2004;
Radostits et al. 2007), but in some cases, lesions were ob-
served in other organs as a result of circulatory shock
(Collett and Mogg 2004; Brown et al. 2007; Radostits et al.
2007). BParatyphoid granules^, which confer a Bturkey-egg^
appearance to the liver and kidney, were not observed in our
cases. Such alteration, already observed in diferent host spe-
cies, had not yet been reported in equine salmonelosis (Brown
et al. 2007). On the other hand, adrenal hemorrhage, which
has been consistently associated with circulatory shock (Jones
et al. 1996), was detected in 50% of our cases.
The main histopathological changes observed in the intes-
tine included diffuse necrosis of the mucosa. This may be a
result of bacterial invasion and the subsequent local necrotiz-
ing effects of neutrophil activity (Astorga et al. 2004; Brown
Table 3 Distribution and severity of the histological lesions in the small and large intestines in 10 equines with salmonellosis in southern Brazil
N° Necrosis of
mucosa
Necrosis of crypt
or of villosity
Inflammation of
mucosa
Edema of
submucosa
Thrombus of
submucosa
Pseudomembrane Lymphoid
necrosis
Lymphatic dilation
in the submucosa
SI LI SI LI SI LI SI LI SI LI SI LI SI LI SI LI
1 + +++ − +++ + ++ − − − − − − − +++ − ++
2 − + − ++ − ++ − + − ++ − − +++ +++ − +
3 − +++ − +++ − +++ − +++ − +++ − ++ − +++ − +++
4 ++ − + − +++ − ++ +++ − − +++ − ++ ++ +++ +++
5 ++ − + − +++ − ++ +++ − − +++ − ++ ++ +++ +++
6 − +++ − +++ − +++ − +++ − +++ − ++ − ++ − +++
7 ++ ++ − ++ ++ ++ ++ +++ ++ − ++ + ++ ++ +++ +
8 + +++ + +++ + +++ − + − +++ − ++ ++ +++ ++ ++
9 − +++ − +++ − ++ +++ ++ − +++ − ++ − +++ − ++
10 − ++ − ++ + + − + − − − − ++ ++ − +
Severity: absent (−), light/discrete (+), moderate (++), accentuated /intense (+++)
SI small intestine, LI large intestine
Table 4 Results of the bacterial isolation assays and serovars identified
in 10 equines with salmonellosis in southern Brazil
N° Isolation Quantitative Serovar
1 Yes + NC
2 Yes ++ Typhimurium
3 Yes ++ Anatum
4 NG − −
5 NG − −
6 Yes ++ Anatum
7 Yes ++ Muenster
8 Yes +++ Typhimurium
9 NG − −
10 Yes + NC
Quantitative: light/discrete (+), moderate (++), accentuated /intense (+++)
NG no growth in 72 h, NC not conducted
480 Trop Anim Health Prod (2017) 49:475–482
et al. 2007; Radostits et al. 2007). The inflammatory infiltrate
was composed predominantly of neutrophils, a finding also
observed in enteritis due to Salmonella in other species, such
as bovines and humans (Santos et al. 2003). Apart from these
changes, other manifestations, such as the formation of
pseudomembranes, vascular and inflammatory lesions in the
submucosa, and lymphoid depletion in follicles associated
with the gastrointestinal tract and spleen, were similar to those
described by other authors in equine salmonellosis (Roberts
and O’boyle, 1982; Brown et al. 2007).
Isolation is the most reliable method for diagnosing salmo-
nellosis. It may be carried out using intestinal contents, lymph
nodes, the liver, and any other organ that shows signs of in-
fection. Its main advantages are the identification of the agent,
the possibility for conducting antimicrobial susceptibility as-
say and isolate serotyping. However, the need for enrichment
and the use of selective media for growth often makes bacte-
rial isolation difficult. In addition, prior antibiotictreatment
and the time that elapses between death and sample collection
upon necropsy are important variables (Traub-Dargatz and
Besser 2007). Here, of the seven cases from which
Salmonella was isolated, only five serovars were character-
ized. This was possibly due to the occurence of non-
typeable strains which are somatic or flagellar antigen defec-
tive variants that arise as consequence of a mutation or loss of
antigen during sample processing (Herrera-León et al. 2007).
Of the serovars identified, Typhimurium and Anatum have
been associated with an enteric clinical picture in equines
(Smith et al. 1978). S. muenster has been detected only in
asymptomatic equines or in clinical cases of bovines with
lesions in the liver, spleen, and womb with no manifestation
in the intestines (Radke et al. 2002). The lesion caused by this
serovar manifested as hemorrhagic enterocolitis with severe
necrosis and hemorrhage in Peyer’s patches, which is different
from the other two serovars, which caused mainly necrotizing
typhlocolitis.
Some infectious diseases caused by Lawsonia
intracellularis, Listeria monocytogenes, Escherichia coli,
and Rhodococcus equi, as well as non-infectious conditions
(triggered by the use of nonsteroidal anti-inflammatory
drugs), produce clinical signs and intestinal lesions similar
to those observed in salmonellosis, which is the main differ-
ential diagnosis associated with pathological findings from
infections by Clostridium difficile and Clostridium
perfringens type C (Diab et al. 2012; Uzal et al. 2012).
However, some pathological changes and epidemiological
characteristics differentiate these infections. In C. difficile
infections, it has been hypothesized that the distribution of
lesions varies with the age of the animal, and the small
intestine is invariably affected in animals younger than 1 year
of age (Keel and Songer 2006; Uzal et al. 2012; Diab et al.
2013). In the present study, lesions in the small intestine
were observed only in adult animals. In salmonellosis,
intestinal lesions do not follow a strict distribution pattern.
These changes may be located in the large intestine, absent
from the small intestine, or found in different sites in the
organs. This disparity was observed in the present study and
may be explained in light of the severity and/or time that
elapsed between the clinical manifestation and death
(Roberts and O’boyle, 1982; Roth 1988; Radostits et al.
2007). The results of bacterial isolation (and epidemiological
factors that differed from those commonly reported) ruled
out infection by Clostridium sp. in the cases reported here.
Moreover, all cases presented with positive intralesion label-
ing in IHC for Salmonella sp. In spite of the fact that E. coli
was isolated in two of the three cases in which Salmonella
sp. was not detected, the macroscopic and histological le-
sions observed are different from those reported in the liter-
ature (Holland et al. 1996).
IHC has been used in the diagnosis of salmonellosis in
swine, ovines, poultry, and equines (Patterson-Kane et al.
2001; Dagleish et al. 2010; Watanabe et al. 2011). This assay
may be used as an alternative when bacterial isolation is not
feasible. Another aspect that underlines the reliability of IHC
is the fact that immunoreaction occurs in the foci of lesions,
mostly on the surface of the mucosa as well as inside macro-
phages and lymphoid tissue. These findings are in accordance
with the invasion of Salmonella sp. in the intestine, which
occurs both through epithelial cells, from the apical region
of villi, and M cells located in Peyer’s patches (Schauser
et al. 2004).
Salmonellosis is one of the most important causes of
enteritis in horses. It is characterized by necrotizing or
fibrin-hemorrhagic enteritis. Knowing the factors associated
with the manifestations of the disease and the macroscopic
and microscopic changes, as well as the use of complemen-
tary assays, facilitates reaching a reliable diagnosis. In spite
of the recent advancements in the knowledge about salmo-
nellosis, the disease may manifest in different forms, as
observed with the serovar Muenster. For this reason, this
subject deserves further investigation. In the diagnosis of
salmonellosis, IHC of the histological lesion is a key tool
when bacterial isolation is not feasible. Improving the di-
agnosis techniques is important because the number of
equine salmonellosis cases of unknown origin is high.
Acknowledgements This work was funded by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Brazil.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Statement of animal rights The manuscript does not contain clinical
studies or patient data.
Trop Anim Health Prod (2017) 49:475–482 481
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482 Trop Anim Health Prod (2017) 49:475–482
	Equine salmonellosis in southern Brazil
	Abstract
	Introduction
	Materials and methods
	Immunohistochemistry
	Bacteriological examination
	Results
	Gross lesions
	Microscopic lesions
	Immunohistochemistry
	Bacterial isolates
	Discussion
	References