Extranodal γδ-T-cell lymphoma in a dog with leishmaniasis

Abstract: An 8-year-old intact male mongrel dog with alopecia and weight loss was referred to the Veterinary Faculty of Naples. The dog had pale mucous membranes, enlarged prescapular lymph nodes, and splenomegaly. Laboratory abnormalities included anemia, thrombocytopenia, and hyperglobulinemia. Bone marrow aspirate smears contained numerous Leishmania amastigotes and an immunofluorescent antibody titer was strongly positive (1:1280) for leishmaniasis. The dog was treated with a combination of meglumine antimoniate and allopurinol for 60 days and showed clinical improvement. Two months after the end of treatment the dog was again referred because of relapse of leishmaniasis and the presence of a firm subcutaneous mass on the medial right thigh. Based on cytologic examination of fine needle aspirates of the mass, a diagnosis of large-cell lymphoma was made. Flow cytometry of tumor cells revealed γδ-T-cell lymphoma with a CD5+, CD3+, TCRγδ+, CD4−, CD8−, CD45RA+ immunophenotype. Using nested PCR, amastigotes were not detected in the neoplastic tissue. An association between leishmaniasis and hematopoietic tumors has been described rarely. γδ-T cells may be involved in the host response to this parasite, and prolonged antigenic stimulation and chronic immunosuppression (typical of leishmaniasis) play a crucial role in the etiopathogenesis of T-cell lymphoma.     

Cerebrospinal fluid from a dog with neurologic collapse

A 3-year-old Staffordshire Terrier was presented to the Texas Veterinary Medical Center with a short progressive history of anorexia, weight loss, and weakness that had progressed to ataxia and collapse with empirical treatment. The dog was tetraparetic and obtunded. Results of a complete neurologic evaluation were consistent with severe, multifocal to diffuse disease involving the forebrain, spinal cord, and brainstem. Cerebrospinal fluid, obtained via cerebellomedullary cisternal puncture, was highly cellular and contained large atypical round cells with small numbers of nondegenerate neutrophils and large mononuclear cells. Rare eosinophils and small lymphocytes were noted. The atypical round cells were approximately 15–25 μm in diameter with a single nucleus set in a small amount of cytoplasm. The nuclei were typically round to slightly ovoid; however, occasional notched, lobulated, and reniform nuclei were observed. These cells were interpreted as malignant lymphocytes. Owing to a grave prognosis, the animal was euthanized and a necropsy was performed. No gross lesions were found in the central nervous system. Multiple sections of cerebellum, medulla, and spinal cord contained a diffuse neoplastic infiltrate that was predominantly meningeal with rare superficial neuropil invasion. The neoplastic cells were arranged in sheets, cords, and rosettes. Immunohistochemical staining for vimentin, pancytokeratin, CD3, CD79a, synaptophysin, S-100, and neuronspecific enolase was negative; glial fibrillary acidic protein (GFAP) staining was equivocal. Based on histologic findings, a diagnosis of medulloblastoma was made. This case documents the rare occurrence of a canine medulloblastoma and illustrates the difficulty in distinguishing between some embryonal brain tumors and lymphoma.     

Cerebrospinal fluid from a dog with hind limb ataxia

A 9-year-old spayed female German Shepherd dog with a history of orthopedic disease was presented to the North Carolina State University Veterinary Teaching Hospital for evaluation of recent, progressive, bilateral, hindlimb ataxia. Analysis of cisternal and lumbar cerebrospinal fluid (CSF) samples revealed normal total nucleated cell counts and a mild increase in protein concentration in the lumbar sample. In cytocentrifuged specimens of both CSF samples, aggregates of refractile, angular to irregular, pale blue to colorless, crystalline material were observed in the background. Some of the material appeared birefringent under polarized light. Differentials for the material included contrast agent, epidural anesthetics or other pharmacologic agents, or artifact introduced through sample processing, collection, or handling. Based on investigation of clinical and laboratory processes it was determined that tubes used to collect CSF in the hospital recently had been changed from additive-free glass tubes to silica-coated shatter-resistant plastic tubes (BD Vacutainer Plus serum tubes, silicone-coated, Becton Dickinson). A cytocentrifuged preparation of saline placed in a silica-coated tube contained crystalline material identical to that observed in the CSF samples; saline placed in an additive-free glass tube contained no material. In this case, we document the microscopic appearance of highly concentrated silica particles in cytocentrifuged preparations of CSF and underscore the importance of recognizing and identifying this artifact in cytologic preparations.     

Cerebrospinal fluid from a 6-year-old dog with severe neck pain

A 6-year-old, intact female, Labrador Retriever/Terrier cross was presented to the University Veterinary Hospital, University College Dublin with a 3-week history of therapy-resistant cervical pain and intermittent fever. Physical examination findings included marked cervical pain resulting in neck extension and vocalization. Examination of the CSF revealed mild pleocytosis (total nucleated cells = 0.009 x 10(9)/L, reference interval <0.005 x 10(9)/L). Cytocentrifuged preparations of the CSF were of low cellularity, containing predominantly macrophages and occasional small lymphocytes. Several small- to medium-sized fragments of a slightly granular, amorphous, eosinophilic substance were observed. The majority of mononuclear cells were located within this material, in small groups of 3-13 cells. The amorphous foamy material stained positive with Luxol fast blue, suggestive of myelin-like material. The dog was euthanized and postmortem examination revealed intervertebral disk protrusion between C2 and C3. Hematoxylin- and Luxol fast blue-stained histopathologic sections of brain and spinal cord revealed only mild hemorrhage. The extracellular material in the CSF of this dog may have been caused by myelin degeneration or leakage of phospholipids from damaged cells. Because no histologic evidence of demyelination was observed with the disk extrusion, the myelin-like material in this case was thought to be the product of phospholipid breakdown from damaged cellular membranes. Three cases of dogs with spinal cord disease and myelin-like material in the CSF have been reported previously. The clinical significance of this finding is still unknown.     

Cerebrospinal fluid from a 10-year-old dog with a single seizure episode

A cerebrospinal fluid sample collected from the cerebellomedullary cistern of a 10-year-old Shetland Sheepdog with a recent history of seizures was submitted for fluid analysis and cytologic examination. Key findings included a total nucleated cell count of 520/microL (reference interval 0-5 cells/microL), with a predominance of mononuclear cells, a protein concentration of 51.8 mg/dL (reference interval 0-35 mg/dL), and a glucose concentration of 44.7 mg/dL (reference interval 52-105 mg/dL). There was marked atypia of the mononuclear cells, with abundant eosinophilic cytoplasm, marked anisocytosis and anisokaryosis, occasional binucleated cells, mitotic figures, and rare erythrophagia. The cytologic interpretation was marked, monocytoid-rich, mixed cell pleocytosis with cellular atypia worrisome for neoplasia. In addition to histiocytic neoplasia, differentials included granulomatous meningoencephalomyelitis, necrotizing meningoencephalitis, and granulomatous inflammation. The dog did not respond to anti-inflammatory and anticonvulsive therapy. At necropsy, a mass involving the meninges and subtending the neuropil of the right temporal lobe of the cerebrum was found. Histologically, the mass was composed of large, bizarre histiocytic cells with multinucleated forms and numerous mitotic figures. Using immunochemistry on cytologic and histologic samples, the pleomorphic histiocytic cells were positive for CD1c, CD11ad, CD45, lysozyme, and vimentin, and were negative for CD3, CD4, CD79a, CD90, and pancytokeratin. These findings supported a diagnosis of primary CNS malignant histiocytosis of dendritic antigen-presenting cell (CD1c+) origin. To our knowledge, this is only the third reported case of primary CNS histiocytic sarcoma in dogs, and the first to demonstrate strong immunochemical evidence for dendritic antigen-presenting cell origin.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Abdominal fluid from a dog

A 2-year-old intact female mixed-breed dog with a 1-month history of lethargy and anorexia was evaluated for abdominal distension and an abdominal mass. The dog's last heat cycle, her third, was 1 month prior to presentation, and no reproductive cycle abnormalities were noted at any time. Hematologic and serum biochemical abnormalities were consistent with hemorrhage and inflammation. Ultrasonographic examination confirmed a large midabdominal mass and a moderate amount of abdominal fluid. Cytologically, the fluid showed evidence of pyogranulomatous inflammation, hemorrhage, and mesothelial reactivity, as well as ciliated columnar cells and free cilia that were interpreted as likely of oviductal origin. The mass was removed surgically, and the histopathologic interpretation was oviductal hamartoma with marked stroma formation and acute hemorrhage. To the authors' knowledge, this is the first reported case of oviductal hamartoma in any species and the first reported case detailing the finding of ciliated columnar epithelial cells in the abdominal fluid of a dog. Ciliated columnar epithelial cells in abdominal fluid should be considered indicative of a likely underlying oviductal lesion.     

Combination Allopurinol and Antimony Treatment versus Antimony Alone and Allopurinol Alone in the Treatment of Canine Leishmaniasis (96 Cases)

The aim of the present study was to evaluate the long-term clinical outcome for dogs with leishmaniasis that were treated with 3 different protocols: combined treatment with antimony and allopurinol, antimony alone, or allopurinol alone. Ninety-six dogs included in this study were determined to have leishmaniasis on the basis of (1) clinical features, (2) identification of the parasite in smears of lymph node, bone marrow aspirates, or skin biopsies, and (3) specific immunofluorescent assay. Three groups of dogs were defined: 45 dogs (group 1) were treated with antimony (100 mg/kg s.c. q24h) given concurrently for 1 month with allopurinol (15 mg/kg p.o. q12h), and then allopurinol alone for 8 months at the same dosage; 40 dogs (group 2) were treated with antimony alone according to the manufacturer's instructions (200 mg/kg s.c. q24h at 2-day intervals for 3-6 months); and 11 dogs (group 3) were treated with allopurinol alone (15 mg/kg p.o. q12h for 1-20 months). Information concerning signalment, history, physical examination findings, serologic testing and number of dogs becoming seronegative, outcome for each treated dog (clinical cure versus failure), and long-term survival were recorded. The numbers of the clinical cures versus failures were significantly different among the 3 groups (chi2 = 17.77, P < .001), between groups 1 and 2 (chi2 = 8.02, P < .01), between groups 2 and 3 (chi2 = 11.00, P < .01), and between groups 1 and 3 (chi2 = 16.52, P < .001). No significant difference between groups 1 and 2 was noted in the type of failure (relapse or death), serologic test results, and number of survival years (chi2 = 2.79, P > .05). The results of the present study indicate that antimony in combination with allopurinol produces better results than antimony alone or allopurinol alone for the treatment of the canine leishmaniasis. With combination treatment, duration of treatment with antimony is shorter and long-term administration of allopurinol is well tolerated.     

Pleural fluid from a dog with marked eosinophilia

A 12-year-old neutered male Shar-Pei was presented to the North Carolina State University Veterinary Teaching Hospital cardiology service with a 2-week history of coughing and a 2-day history of lethargy and anorexia. Pleural effusion and a mediastinal mass were detected with thoracic radiographs. Ten mL of fluid were removed via thoracocentesis, and cytologic examination of the fluid revealed marked eosinophilic inflammation and few atypical mast cells. Mast cell neoplasia was suspected. Aspirates of the mediastinal mass, abdominal lymph nodes, and bone marrow contained similar pleomorphic mast cells and increased numbers of eosinophils. The dog was diagnosed with systemic (visceral) mastocytosis, a rare form of neoplasia in dogs, and was euthanized. These tumors carry a poor to grave prognosis and the etiology is uncertain.     

Evaluation of 65% permethrin spot-on and deltamethrin-impregnated collars for canine Leishmania infantum infection prevention

During the 2004 and 2005 sand fly seasons, we evaluated the efficacy of a 65% spot-on solution of permethrin (Exspot, Schering & Plough) and deltamethrin-impregnated collar (Scalibor, Intervet) in reducing Leishmania infantum infection, in a canine leishmaniasis (CanL) endemic region (Liguria) in Italy. Immunofluorescent assay (IFA) revealed that three of 120 dogs (2.5%) treated with a 65% spot-on solution of permethrin, as three of 119 dogs (2.5%) treated with deltamethrin-impregnated collar have shown seroconversion after sand fly season. On the contrary, seroconversion was 15% in 188 untreated control dogs. Treatment reduced the risk of infection by 84%. The difference in treated dogs and control ones is highly significant (chi2 = 12.4; P = 0.0004). Our results show that treatment with 65% spot-on solution of permethrin and the deltamethrin-impregnated collar are effective in reducing the risk of acquiring L. infantum infection.     

Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.

Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.

Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used.

Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ～10⁰ parasite mL^?1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.

Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.     

Successful resuscitation from cardiac arrest associated with extradural lidocaine in a dog

BACKGROUND: Extradural lidocaine exerts several adverse effects which are seldom fatal. While cardiac arrest following extradural lidocaine injection has been reported in human beings, it has not hitherto been reported in dogs. OBSERVATIONS: The emergency management of a dog with complete urethral obstruction is described. We intended to perform vaginoscopy and cystostomy under extradural lidocaine anaesthesia, but cardiac asystole occurred a few minutes after injection. Resuscitation was successful. About 20 minutes later cardiac arrest recurred, and was treated successfully. The dog remained hypothermic for approximately 7 hours. Complete recovery without neurological deficit occurred the next day and the dog remained normal for at least 3 months. The probable cause of the problem was cranial lidocaine dispersion causing a drop in cardiac preload and cardiac arrest. The successful neurological outcome was attributed to early diagnosis and effective treatment. Hypothermia may have conferred cerebral protection during ischemia. CONCLUSIONS: Extradural local anaesthetic administration is not without risk and the technique should be tailored to individual animals. Constant monitoring is required to detect potentially fatal complications and increase the likelihood of successful outcome.     

Liver aspirate from a Shar Pei dog

A 5-year-old, neutered male, Shar Pei dog was presented with weight loss, anorexia, lethargy, stranguria, and distal limb edema. Clinicopathologic abnormalities included anemia, an inflammatory leukogram, azotemia, icterus, urinary tract infection, and hepatomegaly with a markedly hypoechoic liver. Cytologic findings in a fine-needle aspirate of the liver included large amounts of amorphous, pink, extracellular matrix between hepatocytes. The amorphous material was congophilic using Congo red stain on a hepatic cytology specimen and green birefringent areas were observed under polarized light, confirming the presence of amyloid. The dog was euthanized and a necropsy was done. Histopathologic evaluation using H&E and Congo red staining confirmed amyloid deposits within the liver, kidneys, intestinal vessels, pancreas, and mesenteric ganglia. Immunohistochemical staining of liver and kidney sections using anti-AA amyloid and anti-P component antibodies confirmed the presence of AA amyloid. In this case, we demonstrated that Congo red staining and polarized light microscopy are a useful diagnostic technique in cytologic specimens of suitable thickness for confirming the presence of amyloid.     

Calcospherite-like bodies and caseous necrosis in tracheal mucus from a dog with tuberculosis

A 3-year-old Wirehaired Fox Terrier was presented to the University Veterinary Hospital, University College Dublin, for evaluation of chronic cough of 8-months duration. Bronchoscopy showed a severely dilated collapsed left principal bronchus filled with highly viscous white mucus. Cytologically, globular lipid-like material and round concentrically laminated crystalline structures were evident within the proteinaceous mucus. These findings resembled the calcospherites and granular caseous debris often observed in human tuberculous patients. A Ziehl-Neelsen-stained cytocentrifuged preparation of material obtained by bronchoalveolar lavage revealed a few acid-fast rods within macrophages, suggestive of tuberculosis. At necropsy, granulomas with caseous necrosis were present in the lung parenchyma, bronchial and mediastinal lymph nodes, liver, pancreas, and mesentery. Granulomas were adherent to both kidney capsules and to the diaphragm. Histologically, there was evidence of mild calcification within caseous granulomas, which was confirmed by von Kossa's stain. Using Ziehl-Neelsen stain, acid-fast rods were identified within granulomas; bacterial culture was positive for Mycobacterium bovis. The cytologic findings in this case have not been reported previously in dogs and demonstrate a possible correlation between tuberculosis and calcospherite-like bodies with caseous, globular material in bronchial mucus, similar to that described in human patients.