Trop Anim Health Prod DOI 10.1007/s11250-016-1216-1

REGULAR ARTICLES

Equine salmonellosis in southern Brazil Gregory Duarte Juffo 1 & Daniele Mariath Bassuino 1 & Danilo Carloto Gomes 2 & Fabiana Wurster 1 & Caroline Pissetti 3 & Saulo Petinatti Pavarini 1 & David Driemeier 1

Received: 15 February 2016 / Accepted: 16 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 important 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 presents ten confirmed salmonellosis cases in equines in southern Brazil. Six were adult animals with stress factors preceding the disease, while four were foals, three of which presented 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 characteristic microscopic lesion was mucosa necrosis, which is often accompanied by fibrin deposition, followed by necrosis of follicular centers and vascular changes. Bacterial isolation revealed seven isolates. Five were serotyped, and the

* David Driemeier [email protected] 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

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 exposed 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 estimated that the horse population comprises five million individuals 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 acquired equine salmonellosis cases in southern Brazil and discusses the applicability of immunohistochemistry (IHC) to detect the presence of Salmonella sp.

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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: presence 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 nervous 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 sections were mounted on positive slides (Easy-Path®, Erviegas, SP, Brazil). Endogenous peroxidase blocking Table 1 Identification, history, and clinical signs in 10 equines diagnosed with salmonellosis in southern Brazil

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 confirmed (Watanabe et al. 2011) were prepared simultaneously. Negative controls included sections of intestines of equines that died with no enteric signs and that

Animal

Age

Breed

Gender

History and clinical signs

1

6 months

Mixed

Female

2 3

3 months 5 years

Mangalarga Crioula

Male Male

4 5

7 years 10 years

Mixed Mixed

Male Female

6 7

Adult 8 years

Mixed Crioula

Female Male

8

7 months

Crioula

Female

9

3 years

Mixed

Male

10

40 days

Mixed

Female

Three foals, in the same farm, presented with fetid, yellowish diarrhea, hyperthermia, prostration, and anorexia, and this animal died within 24 h. Animal found dead with no clinical signs. 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. Equines 4 and 5 were from the same farm and would 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. Colic. Laparostomy. Death 6 h after surgery. 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. 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. 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. 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.

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presented with enteric lesions (incubated in PBS, replacing the primary antibody).

Results Gross lesions

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) supplemented with 5% ovine blood and incubated at 35 ± 2 °C for 24/48 h. Samples that exhibited bacterial growth were transferred 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 XyloseLysine-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). 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, brownred 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

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 associated 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 animals, the main finding was observed in this intestinal section. These lesions were characterized by brown-red liquid contents, large levels of free fibrin in the intestinal lumen (Fig. 1d), and the deposition of yellow pseudomembranes in

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the mucosa (2/10) (Fig. 1e). In one animal, the serosa presented 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 hyperemia, hemorrhage, and thrombosis. The inflammatory infiltrate mainly consisted of neutrophils, but macrophages, lymphocytes, plasmocytes, and eosinophils were also present. Diphtheritic membranes associated with mucosa necrosis were observed in the large intestine (4/10) and small intestine (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 necrosis of blood vessels of the submucosa (2/10). The intensity of these lesions is shown in Table 3. Histological lesions in

Table 2 N°

other organs were wide-ranging, including Bparatyphoid nodules^ (4/10) and thrombosis (5/10) in the liver; hemorrhage 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 subendocardial hemorrhage in the heart (3/10); cardiomyocyte necrosis, which at times was mineralized and presented with light mononuclear infiltrate (1/10); microthrombi and severe hemorrhage 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 primary 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 macrophages 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.

Macroscopic changes in the small and large intestines in 10 equines with salmonellosis in southern Brazil

Small intestine

Large intestine

Serosa

Mucosa

Serosa

Mucosa

1 2 3

N.C. N.C. N.C.

N.C. N.C. N.C.

4

Hyperemic with petechiae and ecchymosis Hyperemic with petechiae and ecchymosis. N.C.

Hyperemic, thickened, pseudomembrane. Hyperemia, pseudomembrane.

N.C. N.C. Reddish / hyperemic with focal segmented hemorrhage. Reddish.

Irregular surface, granular aspect Irregular surface, granular aspect Thickened, hyperemic with ulcers and covered by pseudomembrane Edematous

Reddish.

Edematous Thickened and hyperemic with dark red multifocal areas covered with pseudomembrane

7

Hyperemic with petechiae and ecchymosis.

8

N.C.

Hyperemic with fibrin deposition on Peyer’s patches. N.C.

Reddish / hyperemic, with intense focal segmented hemorrhage. Reddish.

N.C.

9

N.C.

N.C.

10

Hyperemic

N.C.

Edematous with ulceration areas covered with pseudomembrane Thickened and hyperemic with button-like ulcer multifocal areas and covered with fibrin Hyperemic, edematous and finely granular

5 6

N.C. no change

N.C.

Reddish / hyperemic with focal segmented hemorrhage. N.C.

Thickened and hyperemic

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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 (hematoxylineosin). 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; streptavidinperoxidase conjugate; AEC chromogen)

Bacterial isolates

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.

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 of S. 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

Trop Anim Health Prod Table 3 N°

Distribution and severity of the histological lesions in the small and large intestines in 10 equines with salmonellosis in southern Brazil

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

Studies of salmonellosis in horses have identified risk factors that increase the likelihood of infection during hospitalization (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 received antibiotic therapy and were undergoing feed restriction or diet change were at greater risk for developing salmonellosis. This is most likely because of alterations in enteric microflora (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 Table 4 Results of the bacterial isolation assays and serovars identified in 10 equines with salmonellosis in southern Brazil N°

Isolation

Quantitative

Serovar

1 2 3 4 5 6 7 8

Yes Yes Yes NG NG Yes Yes Yes

+ ++ ++ − − ++ ++ +++

NC Typhimurium Anatum − − Anatum Muenster Typhimurium

9 10

NG Yes

− +

− NC

Quantitative: light/discrete (+), moderate (++), accentuated /intense (+++) NG no growth in 72 h, NC not conducted

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 observed 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 species, 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 intestine included diffuse necrosis of the mucosa. This may be a result of bacterial invasion and the subsequent local necrotizing effects of neutrophil activity (Astorga et al. 2004; Brown

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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 salmonellosis. It may be carried out using intestinal contents, lymph nodes, the liver, and any other organ that shows signs of infection. Its main advantages are the identification of the agent, the possibility for conducting antimicrobial susceptibility assay and isolate serotyping. However, the need for enrichment and the use of selective media for growth often makes bacterial isolation difficult. In addition, prior antibiotic treatment 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 characterized. This was possibly due to the occurence of nontypeable strains which are somatic or flagellar antigen defective 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 differential 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 labeling 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 lesions observed are different from those reported in the literature (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 macrophages 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 complementary assays, facilitates reaching a reliable diagnosis. In spite of the recent advancements in the knowledge about salmonellosis, 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 diagnosis 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.

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