Introduction

Canine leishmaniasis (CanL) due to Leishmania infantum is a major global zoonosis potentially fatal to humans and dogs, the latter comprising the main reservoir of infection.¹ It is endemic in more than 70 countries of the world, and it is present in regions of southern Europe (Mediterranean region), Africa, Asia, and South and Central America, and has also been reported in the United States.² CanL presents with a broad spectrum of clinical signs and with various degrees of severity: classic CanL appears clinically as a chronic wasting disease with anemia, cutaneous lesions, and generalized lymphoadenopathy. Some dogs also show epistaxis, arthritis, keratoconjunctivitis, and uveitis, and, in the later stages of the disease, chronic kidney disease may appear.³ Diarrhea, originating from either the small or large intestine, is another feature, varying from 3–8% to as high as 30%.^4,5 However, most authors consider chronic diarrhea as an unusual clinical presentation of CanL.⁶

We present an atypical case of canine leishmaniasis in which the primary clinical sign was chronic diarrhea of small intestinal origin with none of the other typical clinical features, except for nonspecific ones such as chronic wasting and anemia.

Case Report

A 26-kg, 1-yr-old intact male boxer was referred for assessment because of a 4-wk history of small intestinal diarrhea with increasing volumes of semiformed feces. There was no tenesmus, vomiting, or hematochezia. The major clinical signs were small intestinal diarrhea and poor body condition (score 3/9; ideal weight 5/9). His diet consisted of a commercial hydrolysed protein diet^a, prescribed by the referring veterinarian in an 8-wk dietary trial with minimal improvement. The dog had been regularly vaccinated and dewormed (every 3 mo; 1 tablet/10 kg body weight) with a febantel, praziquantel, and pyrantel pamoate combination product^b. Antidiarrheic therapy (metronidazole 20 mg/kg q 12 hr for 4 wk, and sulfasalazine 20 mg/kg q 8 hr for 2 wk) had been previously administered without success. On physical examination, he appeared bright and alert, with a moderate level of weight loss, although no abnormalities were detected on abdominal palpation and lymph nodes were palpably normal.

Laboratory data (complete blood count, biochemical profile, and urinalysis; see Table 1) at the time of admission showed a mild nonregenerative anemia, hypoproteinemia, and a raised gamma glutamyl transpeptidase, alkaline phosphatase, and cholesterol. Serum cobalamine and folate levels were within the normal range, and no parasites were found on repeated fecal analysis (fresh saline fecal smears and zinc sulfate centrifugal flotation). A Giemsa stained smear of feces was performed, but no white blood cells were observed and the bacterial population was normal. Radiographs of the abdomen did not reveal any abnormalities. The protein/creatinine ratio in urine was within normal limits.

TABLE 1 Results of Hematological and Serum Biochemical Analysis and Urinalysis

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High concentrations of acute-phase proteins (C-reactive protein [CRP], and haptoglobin; Table 1) were observed at admission using the method described by Martínez-Subiela et al.⁷ Because the trypsin-like immunoreactivity test showed a normal value (Table 1), a diagnosis of chronic enteropathy was provisionally made, with unsuccessful dietary exclusion and antibiotic trials. A gastroduodenoscopy and colonoscopy were performed to detect possible changes in the intestinal mucosa and to obtain biopsy specimens.

Patient preparation for endoscopy included 48-hr fasting (solid food), administration of a senna glycosides oral laxative solution^c the previous day (1 mL/kg), and two enemas 12 and 4 hr before examination (sodium phosphate solutions; 250 mL)^d. The animal was examined under general anesthesia, using propofol for induction and isoflurane for maintenance. Routine endoscopic examination was performed with a flexible video endoscope^e, and the gastric and colonic mucosa appeared normal. The duodenal mucosa exhibited mild edema and increased granularity in several zones and appeared friable upon biopsy. The samples were fixed in formalin, embedded in paraffin, cut in 4-μm-thick sections, and stained with hematoxylin and eosin (H-E). At microscopic analysis, a substantial inflammatory reaction was observed in some samples of the duodenal mucosa with a moderate increase in lamina propria lymphocytes, plasma cells, and macrophages, following the classification of Day et al.⁸ Basophilic bodies were seen in small vacuoles in the cytoplasm of numerous macrophages, with morphology suggestive of Leishmania spp. (Figure 1A, B). Immunocytochemistry, using 4-μm sections from formalin-fixed and paraffin-embedded specimens stained by H-E and by the avidin-biotin-peroxidase complex method using a rabbit polyclonal antibody against L. infantum,⁹ was performed and confirmed that the basophilic bodies were amastigotes of Leishmania (Figure 2). Serology confirmed the presence of leishmaniasis, with a significant anti-Leishmania antibody titer (1/640) detected by indirect immunofluorescence. A diagnosis of granulomatous duodenitis associated with Leishmania infection was therefore made, and treatment was commenced using the guidelines of Solano-Gallego et al.¹⁰ Treatment included meglumine antimonite (100 mg/kg subcutaneously q 24 hr) administered concurrently with allopurinol (15 mg/kg per os [PO] q 12 hr) for 30 days. Since we were not sure enough that inflammation could simply be a consequence of the Leishmania infection, treatment for enteritis was administered, and it included prednisone (1 mg/kg PO q 12 hr), metronidazole (20 mg/kg PO q 12 hr) to prevent potential dysbiosis, cyanocobalamin (2 mg subcutaneously once a wk) to prevent its deficiency, omeprazol (0.5 mg/kg PO q 24 hr), and a hydrolysed protein diet^a (dividing feedings into two meals) was initiated. After 3 wk, the prednisone was gradually decreased (50% reductions at 2-wk intervals and alternate-day treatment for a total of 5 mo). The animal was then kept only on the hydrolysed protein diet and allopurinol twice daily.

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Improvement in the health of the dog was observed after commencement of treatment. The patient gained weight, and diarrhea began to disappear approximately 22 days after initiation of treatment. At the end of treatment (after 7 mo), there was a complete resolution of clinical symptoms and an 8-kg weight gain. Repeat endoscopy and biopsy examination (both by histopathology and immunocytochemistry) did not show any Leishmania. During follow-up, a progressive decline of anti-leishmanial antibody titer was also observed, with values of 1/320 after 2 mo and 1/80 after 7 mo.

Discussion

Chronic diarrhea is generally considered an unusual clinical presentation of CanL, and, in an extensive report of 95 cases, chronic diarrhea was never the primary clinical sign.^5,6 It has been reported in dogs affected by leishmaniasis, but in these cases the affected animals showed other systemic signs including anorexia, emaciation, anemia, lymphoadenopathy, and skin lesions.⁵ In our case, most of the typical signs of leishmaniasis (skin lesions, lymphoadenopathy, ocular lesions, etc.) were not present; only those associated with chronic enteritis (diarrhea as the primary clinical sign, and nonspecific ones such as chronic wasting and anemia).

The occurrence of diarrhea has been mostly associated with chronic renal or hepatic failure that may develop in the course of disease.⁵ However, Leishmania organisms can also cause inflammation of the digestive tract in an isolated manner. Two cases of acute fatal hemorrhagic enteritis have been reported, and some cases of chronic colitis have been observed.^11,12 Enteritis may be the result of direct parasitic damage (granulomatous enteritis) or the consequence of renal failure.¹³ In our case, renal function and urine protein/creatinine ratio were normal. Inflammatory lesions in the gastrointestinal tract associated with the presence of amastigotes inside macrophages have been reported in dogs infected experimentally with Leishmania amastigotes, and asymptomatic colitis has been described in naturally infected dogs with L. infantum.^14,15 Other authors have also suggested that the detection of parasites in dogs naturally infected with L. infantum is common and does not necessarily correlate with intestinal disease.^16,17 Endoscopic biopsy in this report showed the presence of granulomatous inflammation of the duodenal mucosa and the presence of Leishmania parasites. L. infantum could therefore be added to the list of infectious or parasitic agents causing chronic enteritis. The differential diagnosis of disorders resembling inflammatory bowel disease (IBD) in dogs include: chronic giardiasis, dietary sensitivity, dysbiosis, lymphangiectasia, lymphoma, pythiosis, functional bowel disorder, histoplasmosis, exocrine pancreatic insufficiency, adenocarcinoma, and stagnant loop.¹⁸

One of the clinical features of CanL is chronic wasting, although it is also a typical sign of chronic enteritis and numerous other conditions. It has been suggested that extensive infiltration of mononuclear cells in the intestines may result in impaired absorption with subsequent emaciation, but wasting could also be a nonspecific constitutional response to the infection.¹⁹

IBD localized to the small intestine was at first suspected in this case, given the chronic clinical signs and by ruling out other known causes of gastrointestinal inflammation. This was done following the Guidelines of the American College of Veterinary Internal Medicine Consensus Statement, using multiple fecal examinations, biochemical profile, dietary and therapeutic trials, and trypsin-like immunoreactivity prior to endoscopy.²⁰ Observation of basophilic bodies in macrophages was suggestive of Leishmania infection, and it was confirmed by immunostaining. Therefore, IBD was not the final diagnosis, as an idiopathic gut inflammation cannot be diagnosed when infectious agents are seen. Immunohistochemical staining is reportedly more sensitive than H-E or Giemsa staining for the detection of Leishmania amastigotes in various tissues.¹² This report emphasizes the importance of biopsy sampling for definitive diagnosis as opposed to treating empirically for presumed IBD, which would have likely been disastrous in this case.

Unusual intestinal localization of Leishmania has been reported in immunocompromised humans, especially in human immunodeficiency virus-infected individuals and in geriatric immunocompetent subjects.²¹ Our dog had not received any treatment with immunosuppressive effects and was quite young.

Findings of laboratory analysis are typical of chronic disease and chronic enteritis. Hyperproteinemia is typically found in dogs with CanL (or hypoproteinemia in animals with protein-losing nephropathy), but in this case the chronic inflammation of the small intestine and associated diarrhea may have induced hypoproteinemia. Total proteins increased with therapy and achieved a normal value after 7 mo, with a gradual increase in albumin. The γ-globulins decreased after that period, probably as a consequence of the remission of the inflammatory process.

The prognosis of leishmaniasis is uncertain, primarily because the parasite has been shown to be extremely resistant to treatment.⁷ It has been proposed that a precise way to monitor the therapeutic effect is serial evaluation of total plasma proteins and serum protein electrophoresis; these sequential values are compared with baseline values obtained before treatment is initiated.¹³ The serological titer alone is not suitable for the evaluation of the response to treatment, as it is not related to the severity of the disease.²² A progressive decrease of anti-leishmanial antibody titer to normal levels was observed in this case after 7 mo of therapy, when clinical remission was obtained, but other monitoring methods were also used.

Low values of CRP were observed during therapy, and haptoglobin levels were increased at the beginning of therapy, probably because of the use of corticosteroids, but haptoglobin decreased at the end of therapy, when no corticosteroids were used and the animal was in clinical remission. The possible use of acute-phase proteins as biomarkers for monitoring treatment of dogs with leishmaniasis has been reported.⁷ On the basis of these results, CRP and haptoglobin could have greater potential as biomarkers for monitoring the response to treatment, compared with the use of analysis of electrophoretograms.⁷ However, monitoring of treatment should not be based on the results for acute-phase proteins alone; it should also be correlated with other clinical and laboratory variables, because other pathologic conditions or administration of some drugs could increase concentrations of acute-phase proteins and mask the response to treatment for a specific dog.⁷ In this case, a significant decrease of acute-phase proteins, γ-globulins, and anti-leishmanial titer during therapy was observed, but also a clinical improvement of the animal, absence of Leishmania amastigotes in biopsy, and a negative immunohistochemistry after treatment.

CanL is endemic and has a high prevalence in southern Spain as well as in other Mediterranean countries, but it is also an important concern in non-endemic countries where imported sick or infected dogs constitute a veterinary and public health problem.¹⁰ Leishmaniasis is occasionally diagnosed in the United States in dogs of any breed imported from southern Europe or South America, where the infection is endemic, and in foxhound kennels, a 9.8% overall seropositivity was observed.²

To our knowledge, there are no published reports of chronic small intestinal diarrhea being the primary clinical sign associated with Leishmania infection in the dog. Furthermore, a positive cause-and-effect relationship could be suspected because of the absence of Leishmania amastigotes after 7-mo therapy with a combination of meglumine antimonite and allopurinol and repeat duodenal biopsy. However, the response to treatment may not have been a direct result of the anti-leishmanial therapy, as common treatments for IBD were administered concurrently. This case shows atypical clinical findings, which could be included in the spectrum of unusual visceral Leishmania presentations and underlines the importance of searching for the parasites in atypical sites. Besides, our findings suggest the inclusion of CanL in the list of differentials of canine chronic enteritis, especially in endemic areas. Confirmation of this hypothesis justifies further studies in symptomatic dogs with small intestinal chronic diarrhea in endemic countries.