Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

In vitro lectin binding to the outer surface of Spirocerca lupi at different life-stages.

Spirocerca lupi is the esophageal nematode of dogs. Early, transient eosinophilia occurs in experimentally infected dogs, but is absent in advanced cases, suggesting that the nematode evades the dog's immune system. Lectins are proteins or glycoproteins of plant or animal origin, binding different saccharides, with varying specificities and avidities, used to characterize surface haptens in plant and animal parasitic helminths. This study investigated the in vitro binding of six lectins (Concanavalin A [ConA], wheat germ agglutinin [WGA], peanut agglutinin [PNA], soybean agglutinin [SBA], Dolichus biflorus agglutinin [DBA] and Ulex earopaeus agglutinin I [UEA]) to the surface of S. lupi nematodes at different life stages, the L2 and L3 larvae (dead and alive) and to dead adult worms, with negative controls, with and without addition of the six respective inhibitory sugar haptens. Con A moderately bound to surfaces of both live and frozen L3, to the stoma and excretory pores of adult worms, and to the outer surface nematode's eggs, within a female worm, but not to L2. PNA bound only to stoma and excretory pores surfaces in both frozen and live L3. WGA bound strongly to the outer surfaces of live and dead L2 and L3, which resulted in molting of live larvae. These results suggest that the nematode's surface content change during its development. Such changes may play roles in the nematode's interactions with the intermediate and definitive hosts' tissues, and in its ability to evade the immune response, its long survival within the host, and even induce neoplastic transformation.

Evaluation of hemostatic abnormalities in canine spirocercosis and its association with systemic inflammation.

BACKGROUND: Canine spirocercosis is caused by the nematode Spirocerca lupi and is characterized by esophageal fibro-inflammatory nodules that may undergo neoplastic transformation. No sensitive and specific laboratory assays other than histopathology have been reported to differentiate non-neoplastic from neoplastic disease. HYPOTHESIS/OBJECTIVES: Dogs with spirocercosis will have evidence of hypercoagulability based on thromboelastography (TEG)-derived maximal amplitude (MA); increased MA will be correlated with increased acute phase protein (APP) concentrations (C-reactive protein [CRP] and fibrinogen); increased MA and APPs will be exacerbated with neoplastic spirocercosis. ANIMALS: Thirty-nine client-owned dogs with naturally occurring spirocercosis and 15 sex-matched healthy controls. METHODS: A prospective comparative study evaluating TEG, activated partial thromboplastin time, prothrombin time, antithrombin (AT) activity, platelet count and D-dimer concentration, and APPs of dogs with non-neoplastic (n = 24) and neoplastic (n = 15) spirocercosis compared to control dogs. RESULTS: Median MA was significantly increased in the non-neoplastic group (P < .01) and neoplastic group (P < .01) compared to the controls. Both APPs were significantly increased in the neoplastic group compared to the non-neoplastic and control groups. MA was strongly correlated with fibrinogen (r = 0.85, P < .001) and CRP (r = 0.73, P < .001). An MA >76 mm provided 96% specificity and 73% sensitivity for differentiation of disease state. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine spirocercosis is associated with increased TEG variables, MA and α, and decreased AT activity, which may indicate a hypercoagulable state seemingly more severe with neoplastic transformation. MA was correlated with APP in dogs with spirocercosis and can be used as an adjunctive test to support the suspicion of neoplastic transformation.

Vascular endothelial growth factor concentrations in dogs with spirocercosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor associated with tumor development. Spirocerca lupi is a nematode of canids that induces an esophageal nodule that progresses to a sarcoma in 25% of cases. Determination of neoplastic transformation is challenging and usually based on endoscopy-guided biopsies under general anesthesia, an expensive procedure that often yields nondiagnostic, necrotic samples. HYPOTHESIS: Circulatory VEGF concentrations are increased in dogs with neoplastic spirocercosis and can distinguish between dogs with neoplastic and nonneoplastic disease. ANIMALS: A total of 24 client-owned dogs, 9 nonneoplastic, 9 neoplastic, and 6 controls. METHODS: Case-control study. Plasma and serum VEGF concentrations at the time of diagnosis were compared with those of healthy controls. Measurement of VEGF was performed using a canine-specific ELISA. Kruskal-Wallis and Dunn's tests were used for statistical analysis with significance set at P < .05. RESULTS: The median plasma VEGF concentrations of dogs with neoplastic spirocercosis were 629 pg/mL (range, 282-2,366) higher than both the nonneoplastic (<39.5 pg/mL; range, <39.5-716) and control dogs (<39.5 pg/mL; all values, <39.5; P = .0003). The median serum VEGF concentration of the neoplastic dogs was 69 pg/mL (range, <39.5-212) higher than the nonneoplastic (<39.5 pg/mL; range, <39.5-44.13) and control dogs (<39.5 pg/mL; all values, <39.5; P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Both plasma and serum VEGF concentrations can be used to differentiate nonneoplastic and neoplastic spirocercosis. The role of VEGF in neoplastic transformation of S. lupi-induced nodules and the potential utility of anti-VEGF drugs in spirocercosis-induced sarcoma warrant further investigation.

Plasma IL-8 concentrations are increased in dogs with spirocercosis.

The nematode Spirocerca lupi (S. lupi) induces sarcoma in the dog oesophagus in about 25% of cases. The aim of this study was to compare the differences in the cytokine milieu between dogs with neoplastic (n=29) and non-neoplastic disease (n=49) and age- and gender-matched healthy controls (n=25). We measured IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, GM-CSF and MCP-1 in a specific canine multiplex immunoassay kit. Cytokine concentrations were compared between the different groups using the Kruskal-Wallis test followed by Dunn's test. Only IL-8 and IL-18 showed significant differences in their plasma concentration among the three groups. Kruskal-Wallis test revealed a significant (p=0.001) difference in IL-8 concentration between the neoplastic group (634pg/ml), the non-neoplastic (429 pg/ml) and the control groups (150 pg/ml). Post-test analysis revealed a significance difference between the two S. lupi groups and the control group (p<0.01). The highest IL-18 concentration was found in the non-neoplastic group (53 pg/ml), followed by the control group (46 pg/ml) and finally the neoplastic group (33 pg/ml). IL-18 concentrations were significantly higher in the non-neoplastic group than in the neoplastic group (p=0.05). The increased IL-8 in the spirocercosis groups is consistent with the neutrophilic infiltrate in spirocercosis lesions and in those of other inflammatory-induced neoplasias such as Barret's oesophagus and Helicobacter gastritis. IL-18 showed negative regulatory effect in several worm infections and it is possible that it plays the same role in spirocercosis, allowing the worm to evade the host response and to induce neoplastic transformation.

Immunohistochemical characterization of lymphocyte and myeloid cell infiltrates in spirocercosis-induced oesophageal nodules.

Spirocerca lupi is a nematode that infects the dog's oesophagus and promotes the formation of an inflammatory fibroblastic nodule that progresses to sarcoma in approximately 25% of cases. Spirocercosis-associated oesophageal sarcoma is an excellent and under-utilized spontaneous model of parasite-associated malignancy. The inflammatory infiltrate of paraffin-embedded, non-neoplastic oesophageal nodules (n = 46), neoplastic nodules (n = 25) and normal oesophagus (n = 14) was examined by immunohistochemistry using MAC387 (myeloid cells), CD3 (T cells), Pax5 (B cells) and FoxP3 (T regulatory cells) antibodies. Myeloid cells predominated in 70% of nodules, in pockets around the worms' migratory tracts and in necro-ulcerative areas in neoplastic cases. T cells predominated in 23% of cases with a focal or diffuse distribution, in the nodule periphery. No significant differences were observed between neoplastic and non-neoplastic stages. FoxP3+ cells were observed in low numbers, not significantly different from the controls. The inflammation in spirocercosis is characterized by pockets of pus surrounded by organized lymphoid foci. There was no evidence of a local accumulation of FoxP3+ cells, unlike many previous studies that have reported an increase in FoxP3+ T cells in both malignancies and parasite infections. The triggering factor(s) driving the malignant transformation of the spirocercosis-associated chronic inflammatory nodule warrants further investigation.

Anatomical localization of Gnathostoma spinigerum larval antigens by an indirect fulorescent antibody test.

Indirect fluorescent antibody test (IFAT) was performed on sections of Gnathostoma spinigerum advanced third-stage larva with gnathostomiasis, angiostrongyliasis, trichinosis, strongyloidiasis and cysticercosis sera. Positive fluorescence was observed with the first three sera. Fluorescence was associated with the anterior part of the esophagus, surface of the cuticle and cytoplasmic granules of the intestine. Absorption of sera with gnathostome antigen did not elicit fluorescence. The results suggest that substances secreted from the esophagus and intestine constitute antigens in excretory-secretory products of the larva.