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Keywords:

  • horse;
  • intestine;
  • diverticulum;
  • pseudo-diverticulum;
  • lymphoma;
  • peritonitis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Three mature horses presented with progressive weight loss, inappetence, ventral abdominal oedema and lethargy. Two of the animals had intermittent signs of low grade abdominal pain. At presentation, all 3 had hypoalbuminaemia; 2 had hyperfibrinogenaemia and the other had neutrophilia. An oral glucose tolerance test was performed in 2 cases, both of which demonstrated impaired glucose absorption. One pony treated with corticosteroids failed to improve and developed peritonitis and was subjected to euthanasia after 2 weeks. One pony had small intestinal biopsies obtained via a standing flank laparotomy, which revealed a mainly mononuclear cell infiltrate of the mucosa. It failed to respond to treatment with antibiotics and corticosteroids and, after 2 months, developed sternal oedema in addition to the ventral abdominal oedema and peritonitis and was subjected to euthanasia. The remaining pony deteriorated despite symptomatic therapy and was subjected to euthanasia after one week. At post mortem examination, all 3 animals had multifocal lesions of small intestinal wall thickening, mucosal ulceration, pseudodiverticula and enlarged mesenteric lymph nodes. One pony also had a multinodular mass at the root of the mesentery, a mediastinal mass and a lung mass. Histological examination confirmed the presence of lymphoma of the intestinal wall at post mortem examination in each case and immunohistochemistry (including retrospective evaluation of the intestinal biopsies obtained from the pony that underwent a flank laparotomy) indicated that the lymphomas were of T cell origin.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Although rare, lymphoma (lymphosarcoma or malignant lymphoma) is the commonest haematopoietic neoplasm recognised in the horse. Lymphoma can occur in almost any organ, and can therefore be associated with a wide variety of clinical signs. However, several distinct clinicopathological syndromes exist, including multicentric or generalised, alimentary, mediastinal, cutaneous and solitary tumours of extranodal sites (Neufield 1973; Mair and Hillyer 1992; Carlson 1995; Savage 1998; Schneider 2003; Taintor and Schleis 2011). Approximately 19% of horses with lymphoma have the alimentary form, and this type tends to occur in older horses (mean age 16 years in Taylor et al. 2006). The alimentary form of lymphoma most commonly affects the small intestine and can develop as diffuse intestinal infiltrates associated with weight loss and small intestinal malabsorption; focal intestinal or mesenteric masses associated with weight loss and abdominal pain; or combinations of these lesion types (Roberts and Pinsent 1975; Wilson et al. 1985; Platt 1987; Carlson 1995; Hillyer and Mair 1997; East and Savage 1998; Taylor et al. 2006). Diarrhoea may be present, especially if the large intestine is involved (Wiseman et al. 1974; Love et al. 1992; Mitsui et al. 2007; Sanz et al. 2010). The prognosis for horses with multifocal or diffuse lymphoma lesions is grave, although a small number of cases of focal intestinal lymphoma masses, amenable to surgical resection, have been reported (Dabareiner et al. 1996). In many cases of alimentary lymphoma there is involvement of other organs and lymph nodes, which can make the alimentary lymphoma difficult to differentiate from the multicentric form (Wilson et al. 1985; Taylor et al. 2006; Taintor and Schleis 2011).

This report describes 3 cases of alimentary lymphoma in horses characterised by focal areas of severe disruption of all layers of the small intestinal wall with the formation of pseudodiverticula.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Three cases were identified: a 20-year-old Arab gelding (Case 1), a 12-year-old Welsh Section B pony mare (Case 2) and a 30-year-old crossbred pony mare (Case 3). Case 1 had been diagnosed 2 years previously with septic peritonitis. Medical treatment at that time had failed to resolve the peritonitis and the horse underwent an exploratory laparotomy, which identified a multilocular mesenteric abscess adherent to the distal jejunum with associated mesenteric adhesions to adjacent segments of the jejunum. The abscess and a 1.25 m length of jejunum were resected and an end-to-end anastomosis performed. No other significant lesions were identified at the time of the surgery and the horse made an uneventful recovery. Streptococcus equi ssp. zooepidemicus was cultured from the abscess. Serosal inflammation and mesenteric adhesions were identified grossly in the resected jejunum but histological examination of the resected intestine was not performed. In Case 2, intestinal biopsies were collected at laparotomy, following an oral glucose tolerance test 2 months priorto euthanasia. All 3 animals were subjected to euthanasia anda post mortem examination was performed within 1 h of death in all cases.

Biopsy samples (Case 2), a sample of the small intestine at the site of a pseudodiverticulum (Cases 1–3) and mesenteric, mediastinal and lung masses (Case 2) were fixed in 10% neutral buffered formalin, routinely processed to paraffin wax, sectioned at 4 µm and stained with haematoxylin and eosin (HE). In addition, the biopsy samples and a sample of a pseudodiverticulum from each case were examined immunohistochemically to phenotype the lymphomas further (immunohistochemistry was not performed on the biopsy samples at the time of sampling, but was performed retrospectively following euthanasia). Briefly, simultaneous antigen retrieval and de-waxing was performed at pH 9 (Target retrieval solution, High pH)1 for 20 min at 100°C (PT-Link machine)1. Endogenous peroxidase staining was quenched with H2O2 and the sections incubated with mouse monoclonal antibodies against B-cell antigen receptor complex (1:400 dilution, CD79a)1 and a rabbit polyclonal antibody against pan T-lymphocytes marker (1:400 dilution, CD3)1 for 30 min at room temperature. Antibody staining was detected and developed (EnVision-Flex kit)1 with diaminobenzidine. Positive controls were lymph nodes. Negative controls were performed by omitting the primary antibody.

Clinical findings

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

All 3 cases presented with a history of rapid weight loss over periods of 2–10 weeks, inappetence, ventral abdominal oedema and lethargy. Two animals (Cases 1 and 3) showed intermittent signs of low grade abdominal pain (turning to look at the flanks, periods of sternal or lateral recumbency). None of the horses had diarrhoea. Results of initial haematological and serum biochemical profiles at admission are summarised in Table 1. One pony (Case 3) had a neutrophilia, 2 animals (Cases 1 and 2) had hypoproteinaemia and hyperfibrinogenaemia and all 3 cases had hypoalbuminaemia.

Table 1. Results of haematological and serum biochemical profiles at admission
 Case 1Case 2Case 3Reference range
Packed cell volume %27.744.040.632.0–46.0
Haemoglobin g/l102166120110–170
Red blood cells ×1012/l 8.638.446.0–10.0
White blood cells ×109/l4.39.19.16.0–10.0
Granulocytes ×109/l2.74.57.31.8–6.8
Mononuclear cells ×109/l1.64.61.81.5–7.6
Platelets ×1012/l193119188100–300
Total protein g/l43445753–76
Albumin g/l13121423–37
Globulin g/l30204320–44
Plasma fibrinogen g/l5.04.62.90–4.0

Routine physical examinations identified only poor body condition and ventral abdominal oedema. Transcutaneous, abdominal ultrasound examination revealed evidence of small intestinal wall thickening (>0.5 cm) in Cases 1 and 2. Ultrasonography in Case 3 revealed an ill-defined focal lesion of heterogeneous echogenicity adjacent to a loop of small intestine in the left cranioventral abdomen. An oral glucose tolerance test (Roberts and Hill 1973) was performed in Cases 1 and 2, which revealed evidence of impaired absorption in both horses (maximum increase in plasma glucose concentration above baseline 24 and 32%, respectively). Abdominocentesis was performed in 2 animals (Cases 1 and 2) and the results are summarised in Table 2.

Table 2. Results of initial peritoneal fluid analysis
 Case 1Case 2Case 3Reference range
Nucleated cells ×109/l1.93.3Not performed<5.0
% Neutrophils847820–90
% Mononuclear cells16225–50
Total protein g/l4953<25

Case 1 was treated with systemic dexamethasone (Dexadreson)2 for 30 days (0.1 mg/kg bwt i.v. q. 24 h for 3 days followed by 0.08 mg/kg bwt i.v. q. 24 h for a further 6 days followed by 0.06 mg/kg bwt q. 24 h for a further 21 days) with no observable clinical improvement. A repeat oral glucose tolerance test at the termination of the treatment gave a similar result to the earlier examination (18% [compared with 24%] maximum increase in plasma glucose concentration above baseline). Abdominal paracentesis performed at the termination of treatment yielded turbid peritoneal fluid with a total nucleated cell count of 13.6 × 109/l and total protein concentration of 18 g/l; cytological examination revealed 88% neutrophils and 12% mononuclear/mesothelial cells with no identifiable bacteria or neoplastic cells. On the basis of the continued clinical deterioration, lack of response to systemic dexamethasone therapy and development of peritonitis, the horse was subjected to euthanasia on humane grounds.

In Case 2, following the abnormal oral glucose tolerance test results, a standing flank laparotomy was performed to obtain full-thickness small intestinal wall biopsies. The jejunum and ileum appeared grossly normal but felt thickened. Biopsies were taken from 3 separate sites (proximal and mid jejunum and ileum). Histopathological examination revealed stunting of the villi at all sites (Fig 1), with, in one section, focal loss of villous and crypt architecture leaving a flattened area of mucosa covered by a single layer of epithelium with focal erosion of the surface and a mixed mononuclear cell infiltrate of the lamina propria. At the time, the changes were considered to be inflammatory without evidence of neoplasia, and immunohistochemistry was not performed. The pony was treated with a 5 day course of antibiotics (benzylpenicillin sodium [Crystapen2, 22,000 iu/kg bwt i.v. q. 8 h], gentamicin sulphate [Genta Equine 10%3 6.6 mg/kg bwt i.v. q. 24 h]) prior to discharge. She was then maintained on oral trimethoprim-sulphonamides (25 mg sulpadiazine and 5 mg trimethoprim [Norodine]4/kg bwt per os q. 24 h)5 for 2 weeks prior to starting oral prednisolone (Prednicare5 1 mg/kg bwt q. 24 h). Over the next 2 months the pony's clinical condition continued to deteriorate and she developed dyspnoea, and pectoral and sternal oedema in addition to the ventral abdominal oedema. A sample of peritoneal fluid obtained at this time was cloudy and turbid, with a total nucleated cell count of 21.5 × 109/l and total protein concentration of 44 g/l; cytological examination revealed 97% neutrophils and 3% mononuclear/mesothelial cells with no identifiable bacteria or neoplastic cells. She was subjected to euthanasia at the owner's request without further evaluation.

image

Figure 1. Case 2, jejunal biopsy. Blunted villi with mononuclear cell infiltrate of lamina propria. HE, bar 100 µm. Inset: CD3-positive cells in epthelium and lamina propria. Immunostain CD3 (performed retrospectively after euthanasia), bar 100 µm.

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Case 3 was managed symptomatically for one week by increasing the energy content of the diet, oral multivitamin/mineral supplements and oral pre-biotiocs. At re-examination, the pony had continued to lose weight and was more depressed and inappetent. Repeat haematological and serum biochemical evaluations revealed leucocytosis and neutrophilia (white blood cells 14.2 × 109/l; polymorphonuclear neutrophils 12.3 × 109/l); increased plasma fibrinogen concentration (5.3 g/l) and elevated alkaline phosphatase concentration (276 u/l; reference range 20–180 u/l). Other biochemical indices were similar to the previous results. At the owner's request the pony was subjected to euthanasia without further evaluation.

Pathological findings

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Post mortem findings

In each case, multiple focal areas of small intestinal thickening and multiple focal areas of outpouching (pseudodiverticula) (ranging from 5 to over 25) (Fig 2) of the small intestinal wall were observed. These ranged in size from approximately 1–10 cm in diameter and were most numerous at the anti-mesenteric border of the intestine. The pseudodiverticula were found scattered along the entire length of the small intestine but were most numerous in the distal jejunum and ileum. The mucosal surface was patchily hyperaemic and the normal corrugated surface was focally replaced by multiple irregular flattened plaques. In the areas of pseudodiverticulum formation, the mucosa was deeply ulcerated and in many lesions the surface was covered by malodorous, necrotic and diphtheritic tissue. The large intestine was not grossly affected. Mesenteric lymph nodes were moderately enlarged in all 3 horses and in Case 2 there was a multinodular mass at the root of the mesentery, a mediastinal mass, approximately 12 cm in diameter and a smaller mass (approximately 5 cm in diameter) in the right diaphragmatic lung lobe. On cut surfaces, the lymph nodes and additional masses in Case 2 had a homogeneous consistency of pale cream tissue. In Case 1, there was a generalised peritonitis, with a full thickness perforation of one of the pseudodiverticula and leakage of bowel contents into the abdominal cavity. In Case 2, mild diffuse peritonitis and extensive adhesions of the greater omentum to the serosal surface of several pseudodiverticula was observed.

image

Figure 2. Case 3, jejunal pseudodiverticulum. Inset: opened intestine.

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Histopathological findings

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Histological examination of tissue samples from a small intestinal pseudodiverticulum of all 3 horses showed similar pathological changes. These consisted of mucosal necrosis and infiltration of neoplastic round cells in the mucosa and submucosa (Fig 3) and extending through the muscularis to the serosal surface. The mesenteric, mediastinal and pulmonary masses in Case 2 had a similar histological appearance with sheets of round neoplastic cells. The histological features in all 3 cases were considered to be typical of lymphoma.

image

Figure 3. Case 2, jejunal pseudodiverticulum. Mucosal necrosis and neoplastic lymphoid cell infiltrate of mucosa, submucosa and muscularis. HE, bar 200 µm. Inset: CD3-positive cells in submucosa. Immunostain CD3, bar 100 µm.

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Immunohistochemical examination of samples obtained from intestinal pseudodiverticula in all 3 cases revealed that neoplastic cells were CD3-positive (Fig 3) with only occasional CD79a-positive cells, indicative of T cell lymphoma. Subsequent examination, following euthanasia, of the intestinal biopsies from Case 2, indicated that the mononuclear cells infiltrating the mucosa, including the epithelium, were CD3-positive (Fig 1).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

Three cases of intestinal pseudodiverticula associated with lymphoma in horses were identified. The clinicopathological features of these 3 cases of lymphoma were similar but unusual. Most previous reports of alimentary lymphoma in the horse have described focal intestinal and/or mesenteric masses (usually involving the small intestine and often associated with infiltration and enlargement of mesenteric lymph nodes) or patchy to diffuse mucosal or transmural thickening associated with infiltration of the intestinal wall (Platt 1987). Ulcers of the intestinal mucosa, sometimes with necrotic centres, have been previously reported in association with lymphoma (Roberts and Pinsent 1975; Wilson et al. 1985; Herraez et al. 2001; Pinkerton et al. 2002; Sanz et al. 2010), but, to the authors' knowledge, pseudodiverticula as identified in the 3 horses in the present study do not appear to have been described previously. These lesions were not true diverticula (which involve outpouchings of all layers of the intestinal wall and are usually congenital in origin). Instead, they appeared to be pseudodiverticula (or false diverticula) (Evers 2004), which result from disruption or weakening of the tunica muscularis allowing the other layers of the wall to bulge out as outpouchings. In these horses all layers of the wall, except the serosa, were infiltrated by neoplastic cells. Pseudodiverticula are most commonly reported along the mesenteric border of the intestine (Evers 2004), which is thought to be because this region represents an area of relative weakness associated with perforation of the wall by the intestinal vasculature. In contrast, the pseudodiverticula in these 3 horses occurred predominantly along the anti-mesenteric border, which could relate to the position of the Peyer's patches that may have been the initial site of lymphoma development. In 2 of the horses it appeared that these focally diseased areas had probably allowed leakage of bowel contents with the subsequent development of diffuse peritonitis. Indeed, in one horse (Case 1), a focal perforation of the bowel wall at the level of one of the pseudodiverticula was found at post mortem examination. In Case 2, several areas of adhesions between the greater omentum and the serosal surface of the pseudodiverticula were identified, which suggests a reaction to severe serosal inflammation.

Weight loss and oedema are the commonest presenting signs of lymphoma in horses (Meyer et al. 2006) and were identified in these 3 cases. Likewise, the most common clinicopathological features include hyperfibrinogenaemia and hypoalbuminaemia (Meyer et al. 2006), both of which were also evident in our cases. Neoplastic cells were not identified in either of the horses in which samples of peritoneal fluid were obtained; this is not unusual in cases of alimentary lymphoma since exfoliation of neoplastic cells into the peritoneal fluid rarely occurs (Neufield 1973; Mair and Hillyer 1992; Meyer et al. 2006). In addition to the nonspecific signs of weight loss and oedema, Case 2 developed pectoral and sternal oedema and dyspnoea later in the course of the disease. These additional signs are commonly recognised in horses with mediastinal lymphoma (Mair et al. 1985) and demonstrate the wide range of signs that can occur with lymphoma depending on the distribution of lesions.

The clinical progression of the disease in these 3 horses was rapid following the onset of clinical signs, consistent with previous reports of alimentary lymphoma (Roberts and Pinsent 1975; Wilson et al. 1985; Platt 1987; Carlson 1995; Hillyer and Mair 1997; East and Savage 1998; Taylor et al. 2006). Interestingly, 2 of the horses had had previous surgical exploration of the abdomen at which time evidence of lymphoma or pseudodiverticula had not been identified. In Case 1, an exploratory laparotomy performed under general anaesthesia had been undertaken 2 years prior to the current episode of disease because of unresponsive peritonitis. An abscess associated with the distal jejunum was identified and resected at this time; although no gross evidence of neoplasia was identified, the exact cause of the abscess was not determined and no histopathological examinations were undertaken. It is possible that lymphoma was present in the affected bowel at this time, but in the absence of histopathological examination, this possibility cannot be confirmed or refuted. In Case 2, a standing flank laparotomy was performed 2 months prior to euthanasia to obtain full thickness small intestinal biopsies to investigate the small intestinal malabsorption identified by an oral glucose tolerance test. Although extensive exploration of the abdominal cavity was not undertaken at this time, since for practical reasons only a 10 cm long flank incision was used, there was no evidence of pseudodiverticula of the small intestine. Furthermore, the biopsies from 3 separate levels of the small intestine at the time were not recognised as lymphoma on histological examination. However, subsequent immunohistochemistry revealed T cell lymphoma, which was predominantly epitheliotropic. Two months later, at post mortem examination, multiple areas of neoplasia and pseudodiverticula in the small intestine were found. These findings illustrate the value of immunohistochemistry in cases where the changes are doubtful in routine histological sections. The immunohistochemistry findings in these cases were similar to earlier reports of immunophenotyping of equine lymphomas, suggesting that multicentric, alimentary and mediastinal lymphomas are most frequently of T cell origin (Pinkerton et al. 2002; Meyer et al. 2006; Taylor et al. 2006; de Bruijn et al. 2007; Sanz et al. 2010). Epitheliotropic lymphoma appears to be rare in the horse with only 2 previous reports (Pinkerton et al. 2002; Sanz et al. 2010).

In conclusion, these 3 cases of small intestinal lymphoma of T cell origin caused a rapidly progressing disease characterised by weight loss, inappetence, general depression and, in 2 of the 3 cases, peritonitis, and resulted in multiple pseudodiverticula formation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

We thank colleagues at Bell Equine Veterinary Clinic for clinical assistance, and colleagues at the Comparative Pathology Laboratory, University of Bristol and Bridge Pathology Ltd, Bristol for pathological investigations and expert technical help.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References

1 Dako UK Ltd, Ely, Cambridgeshire, UK.

2 Intervet Schering Plough Animal Health, Milton Keynes, Buckinghamshire, UK.

3 Franklin Pharmaceuticals, Trim, Co. Meath, Ireland.

4 Norbrook Pharmaceuticals, Great Corby, Cumbria, UK.

5 Animalcare Ltd, York, UK.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Clinical findings
  6. Pathological findings
  7. Histopathological findings
  8. Discussion
  9. Conflicts of interest
  10. Source of funding
  11. Acknowledgements
  12. Manufacturers' addresses
  13. References