A compared histopathological study on kidneys and eye bulbs in distinct clinical presentations of canine leishmaniasis by Leishmania infantum.

Canine leishmaniasis (CanL) caused by Leishmania infantum commonly progresses with renal and ophthalmic lesions associated with active systemic disease. As chronic inflammation related to immune complex deposits is a pathophysiological factor in the development of both glomerulonephritis and uveitis, we aimed to evaluate renal and ocular histopathological lesions and analyze whether they were related to each other and the clinical degree of the disease. For that, we evaluated 15 dogs from CanL-endemic areas. L. infantum PCR-positive dogs were studied according to disease severity into two different groups: Group-1 (G1) had data from seven dogs with mild to moderate CanL and no history of treatment, and G2 was formed with eight dogs with severe to terminal disease that had not responded to CanL treatment. Histopathological analysis of kidneys showed higher frequencies and intensities of glomerular basement membrane thickening (p = 0.026), deposits in glomeruli (p = 0.016), epithelial necrosis (p = 0.020), tubular dilatation (p = 0.003) and interstitial fibrosis (p = 0.04) in G2 dogs than in G1 dogs. Surprisingly, the histopathology of eye bulbs showed a higher frequency and intensity of retinitis (p = 0.019) in G1 dogs than in G2 dogs. The comparative analysis showed that there was no correspondence between histopathological findings in kidneys versus eyes in milder or more severe CanL. Our findings suggested that (1) clinically undetectable eye alterations can be more precocious than those in kidneys in the development of CanL, and (2) the lower frequency of eye lesions and higher frequency of renal lesions in dogs with terminal disease even after treatment indicate that therapy may have been effective in reducing CanL-associated ophthalmic disease but not proportionally in reducing kidney disease.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

GATA-3 expression and its correlation with prognostic factors and survival in canine mammary tumors.

Introduction: The transcription factor GATA-3 plays a significant role in mammary gland development and differentiation. Recent studies on human oncology have demonstrated its association with favorable pathologic factors in breast cancer. Canine mammary tumours, proposed as comparative and translational study models, have epidemiological, clinical, biological, and genetic characteristics similar to those of human breast cancers. Methods: Here, we evaluated the frequency of GATA-3 expression in mammary tumors of dogs and its relationship with prognostic factors and survival. Tumor samples were obtained from 40 female dogs and grouped according to histological type into benign tumors (n = 10), carcinoma in mixed tumors (CMTs) (n = 20), and aggressive tumors (n = 10). CMTs were further separated according to histological grade, and data on clinical staging and diagnosis, histopathological grading, and survival rate were collected. Results: GATA-3 and estrogen receptor (ER) expression were higher in benign and well-differentiated carcinomas than in aggressive tumors, which showed greater Ki-67 expression. The expression rate of ER in the studied groups was equivalent to that of GATA-3. We identified a strong positive correlation between GATA-3 and ER expression frequencies and a negative correlation between those of GATA-3 and Ki-67. There were associations between GATA-3 (p < 0.001), Ki-67 (p = 0.003), tumor size (p < 0.001), clinical stage (p = 0.002), lymph node metastasis (p < 0.001), and histological grade (p < 0.001) by univariate survival analysis. The parameters ER (p = 0.015) and GATA-3 (p = 0.005) also influenced survival in a multifactorial manner. Discussion: Kaplan-Meier analysis of survival curves validated our previous findings that dogs with GATA-3 expression in ≥79.4% of cells had significantly higher survival rates (p < 0.001). The performance analysis showed that the expression of GATA-3 in ≥79.4% of cells effectively predicted survival or death in dogs with mammary tumors. Collectively, these results suggest that GATA-3 can be a relevant marker in the study of mammary tumor progression and has potential as a prognosis marker for predicting outcomes in canine mammary tumors.

Clinical evolution of equine leishmaniasis with self-limiting cutaneous disease caused by Leishmania infantum in northeastern Brazil: A case report.

Leishmania infantum infections have long been described in humans and dogs worldwide, but characterization of equine cases remains scarce. We describe the clinical evolution of a natural L. infantum infection to contribute to the diagnostic knowledge and epidemiology of equine leishmaniasis (EL). An auction-acquired four-year-old Mangalarga Marchador mare from Pernambuco state, presented a few subcutaneous nodules on the head and neck upon arrival at the purchaser's stud at Bahia state, in November of 2019. They progressed to multiple ulcerated and non-ulcerated nodules and spread to both right limbs in seven weeks. Hematology revealed anemia, lymphocytosis, monocytosis, and elevated plasma fibrinogen. Histopathology of the biopsied nodules identified a granulomatous dermatitis with macrophages containing Leishmania amastigotes. PCR detected Leishmania in skin lesions, but not in blood or spleen aspirate samples; ITS1 PCR-RFLP and DNA sequencing confirmed L. infantum species. A topical antiseptic and insect-repellent therapy and a monthly follow-up were established. All lesions improved progressively, without specific anti-Leishmania treatment, and 14 months later there was a consistent resolution. This first description of EL by L. infantum in an endemic area is relevant to emphasize the need for epidemiological studies, and to enhance clinicians' awareness for differential diagnosis.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

Pharmacokinetics of Carboplatin in Combination with Low-Dose Cyclophosphamide in Female Dogs with Mammary Carcinoma.

This prospective study aimed to evaluate the effect of metronomic cyclophosphamide on carboplatin's tolerability, efficacy, and pharmacokinetics in dogs with mammary carcinoma. Sixteen female dogs with mammary carcinoma were divided into groups: 300 mg/m2 intravenous (i.v.) carboplatin therapy (G1 = 8) or 300 mg/m2 i.v. carboplatin which was associated with 12.5 mg/m2 oral cyclophosphamide in a metronomic regimen (G2 = 8). The investigated animals underwent a clinical evaluation, a mastectomy, a carboplatin chemotherapy, and serial blood sampling for the pharmacokinetic analysis. The adverse events and survival rates were monitored. A non-compartmental analysis was applied to calculate the pharmacokinetic parameters of carboplatin in the 2nd and 4th chemotherapy cycles. Carboplatin PK showed high interindividual variability with a 10-fold variation in the area under the plasma concentration−time curve (AUC) in G1. The systemic plasma exposure to carboplatin was equivalent in both of the treatments considering the AUC and maximum plasma concentration (Cmax) values. Although the red blood cells (p < 0.0001), platelets (p = 0.0005), total leukocytes (p = 0.0002), and segmented neutrophils (p = 0.0007) were reduced in G2, the survival rate increased (p = 0.0044) when it was compared to G1. In conclusion, adding low daily doses of cyclophosphamide to a carboplatin therapy showed promising outcomes in female dogs with mammary tumors.

Feline Leishmaniasis Caused by Leishmania infantum: Parasite Sequencing, Seropositivity, and Clinical Characterization in an Endemic Area From Brazil.

Zoonotic leishmaniasis caused by Leishmania infantum is a disease of One Health concern since human and animal cases and environmental damage are interconnected. L. infantum has a complex epidemiological cycle with multiple hosts, including mammals-humans, domestic, and wild animals-and arthropod vectors. Knowledge on mammal infections in endemic areas is crucial for developing control strategies. This work aimed to detect and characterize L. infantum infection in domestic cats from areas where human and canine leishmaniasis cases occur. No cases of feline leishmaniasis (FeL) had been previously reported in those areas. Five municipalities from Bahia state were chosen, comprising 2,480.8 km2 with 1,103,866 inhabitants. Ninety domiciliated and/or sheltered cats underwent clinical examination and serology by a rapid reference test recommended by the Brazilian government. Cytology, PCR, and parasite DNA sequencing were performed in bone marrow samples. Rapid tests detected antibodies in 5.6% (5/90) of the cats. Leishmania infantum infection was confirmed in 7.8% (7/90) of the cats by PCR, sequencing, and parasite isolation. Three out of the five municipalities (60%) had infected cats, and PCR positivity varied from 6.9 to 29%. One cat was categorized as harboring active L. infantum infection with amastigote forms in bone marrow smears. No clinical signs were detected at the first clinical exam, but 1 month later the cat developed severe FeL. The cat isolate was grown in culture, typed and its DNA sequence was homologous to the L. infantum reference strain (PP75). In conclusion, cats are potential hosts and may acquire L. infantum in endemic areas where canine and human cases occur. For cats, the need for surveillance, differential diagnosis and clinical care is highly recommended since a fast clinical progression of FeL developed in a subclinical animal. An accurate standardized immunodiagnostic assay for FeL is warranted.

Puppyhood diet as a factor in the development of owner-reported allergy/atopy skin signs in adult dogs in Finland.

BACKGROUND: The increased prevalence of atopic dermatitis (AD) in dogs necessitates research in its disease etiology. OBJECTIVES: To explore the association between puppyhood dietary exposures and prevalence of owner-reported allergy/atopy skin signs (AASS) after the age of 1 year. ANIMALS: Four thousand and twenty-two dogs were eligible, 1158 cases, and 2864 controls. METHODS: This cross-sectional hypothesis-driven observational study was extracted from the DogRisk food frequency questionnaire. Forty-six food items and the ratio of 4 major diet types were tested for their association with AASS incidence later in life. Potential puppyhood dietary risk factors for AASS incidence were specified using binary multivariable logistic regression. The model was adjusted for age and sex. RESULTS: Eating raw tripe (odds ratio, 95% confidence intervals OR, 95% CI = 0.36, 0.16-0.79; P = .01), raw organ meats (OR, 95% CI = 0.23, 0.08-0.67; P = .007), human meal leftovers, and fish oil supplements as well as eating more that 20% of the diet as raw and/or <80% of the diet as dry, in general, were associated with significantly lower AASS incidence in adulthood. In contrast, dogs fed fruits (OR, 95% CI = 2.01, 1.31-3.07; P = .001), mixed-oil supplements, dried animal parts, and dogs that drank from puddles showed significantly higher AASS incidence in adulthood. CONCLUSIONS AND CLINICAL IMPORTANCE: Puppyhood exposure to raw animal-based foods might have a protective influence on AASS incidence in adulthood, while puppyhood exposure to mixed oils, heat processed foods and sugary fruits might be a potential risk factor of AASS incidence later. The study suggests a causal relationship but does not prove it.

Nutritional adjuvants with antioxidant properties in the treatment of canine leishmaniasis.

Clinical improvement of dogs treated for canine leishmaniasis (CanL) requires reducing Leishmania infantum loads, which depend on intracellular oxidant compounds to destroy the parasite. However, oxidative species' excess and antioxidants consumption can culminate in oxidative stress, resulting in increased, widespread inflammation. We aimed to evaluate if early or late addition of nutritional adjuvants (NAs) - omega-3 polyunsaturated fatty acids and B vitamins - to anti-Leishmania drugs (ALDs) in the treatment of CanL would be clinically beneficial. For that, serum biomarkers including oxidative stress parameters were analyzed during 12 months in dogs allocated to two treatment groups: (G1) NAs administered from 30 days prior to the beginning of ALDs; and (G2) NAs administered from 61 days after the beginning of ALDs. Both G1 and G2 continued to receive NAs until the 12th month. The ALDs administered were metronidazole associated with ketoconazole (40 days), followed by allopurinol from day 41 until the 12th month. G1 exhibited superior inflammation control, with reduced globulins (p = 0.025), specific anti-Leishmania immunoglobulins (p = 0.016), total protein (p = 0.031), and an increased serum albumin/globulin ratio (p = 0.033), compared to G2. The early use of NAs associated with ALDs is clinically beneficial in treating dogs with CanL.

Spatial distribution of canine Leishmania infantum infection in a municipality with endemic human leishmaniasis in Eastern Bahia, Brazil.

Efforts to control a zoonotic disease such as visceral leishmaniasis (VL) caused by Leishmania infantum can be successful if they rely on comprehensive data on animal infection. In Bahia state, Brazil, human VL is endemic, yet some areas have no epidemiological data on canine L. infantum infection and canine leishmaniasis (CanL) to date. We aimed to perform an epidemiological study describing the spatial distribution and characterizing canine L. infantum infection in two districts of the municipality of Muritiba, where human cases have occurred. Brazilian official serodiagnostic protocol (ELISA and immunochromatographic tests), PCR and clinical examination were performed in 351 owned dogs. A seroprevalence of 15.7% (55/351) was found, and L. infantum identified in 88.8% (32/36) of PCR tested samples. Spatial distribution of positive dogs indicated infection in both urban and rural districts. There was no association between seropositivity and sex or breed, but dogs older than 2 years were 3.8 times more likely to be seropositive (95% CI 1.57 - 9.18) than younger dogs. Among seropositive dogs, 80% (44/55) had clinical manifestations of CanL: 75% (33/44) presented dermatopathy, 50% (22/44) emaciation, and 29.5% (13/44) ophthalmopathy. This is the first report on canine seroprevalence and natural L. infantum infection in Muritiba, Bahia.

Histopathological Parameters of Canine Bone Marrow in Cell-Block Preparations.

Studies using the cell-block technique for bone marrow (BM) analysis are limited in the veterinary literature. This work assessed whether the histopathological analysis of canine BM was feasible using cell-block cytoinclusions prepared from fine-needle sternal aspirate samples. Eight clinically healthy young-to-middle-aged dogs underwent both fine-needle sternal aspiration for BM cell-block (BM-Cb) processing and iliac-crest BM core biopsy (BM-B). Histopathologic parameters were compared between the 2 methods. There were no statistically significant histopathological differences between hematopoietic tissue areas (P = .6294) in the BM-Cb and BM-B sections, and they had similar microscopic characteristics and microarchitecture. Cellularity and reticulin-fiber staining were equivalent in the BM-Cb and BM-B preparations in 87.5% (7/8) and 100% (8/8) of the sections, respectively. However, the quantitative results of the megakaryocytic series differed between BM-Cb and BM-B in 37.5% (3/8) of the sections, and the myeloid:erythroid (M:E) ratios differed between the 2 methods in 25% (2/8). These preliminary data indicate that cell-block preparations made from sternal fine-needle aspiration samples warrant continued evaluation in a larger number of animals, including those with various diseases affecting the bone marrow.

Leishmania infantum-Derived Glycoinositolphospholipids in the Immunodiagnosis of Subclinically Infected Dogs.

Lipophosphoglycan (LPG), when used as an ELISA target, confers high specificity and sensitivity to the detection of Leishmania infantum antibodies in dogs. Glycoconjugates are economically viable because the yield is very high after extraction/purification. In addition, they are very stable, which allows their use in point-of-care testing without special storage conditions. During the glycoconjugate extraction, a glycoinositolphospholipid (GIPL)-enriched fraction is obtained in similar quantities as LPG. Since GIPLs can be extracted from the same parasite pellet as LPGs, this work aimed to evaluate the immune recognition of GIPLs by Leishmania infantum-infected dogs and its use for canine leishmaniasis (CanL) immunodiagnosis. Like LPG, GIPLs were recognized by sera from L. infantum-infected dogs, but with less sensitivity (83.8%). However, 80% (16/20) of subclinically infected dogs were detected as positive in the assay. Different from LPG, the GIPL-based assay achieved a lower specificity (73.7%) and cross-reactions occurred with T. cruzi and L. braziliensis-infected dogs. Although GIPLs exhibited a similar performance to LPG for subclinically L. infantum-infected dogs, the occurrence of cross-reactivities with other protozoa and a lower sensitivity hinders its use for an immunodiagnostic test. In places where those diseases do not co-exist such as in the Mediterranean region, its use for subclinically dogs could be an alternative.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Targeted Metabolomics With Ultraperformance Liquid Chromatography-Mass Spectrometry (UPLC-MS) Highlights Metabolic Differences in Healthy and Atopic Staffordshire Bull Terriers Fed Two Different Diets, A Pilot Study.

Background: While anecdotal evidence has long claimed that a raw meat-based diet (RMBD) improves the metabolic health of canines, no rigorous scientific study has clarified this issue. Canine atopic dermatitis (CAD) has also been linked to metabolic health, but its relation to diet remains poorly understood. This study investigates whether dietary choice is linked to metabolic health in healthy and CAD-diagnosed canines via targeted serum and urine metabolomic analysis of polar, non-ionic metabolites, as well as whether the underlying CAD condition modulates the response to nutritional intake. Materials and Methods: Serum metabolites of client-owned Staffordshire bull terriers, divided into CAD-diagnosed (n = 14) and healthy (n = 6) cohorts, were studied. Urine metabolites of a subset of the CAD-diagnosed canines (n = 8) were also studied. The canines were split into two cohorts based on diet. The first cohort were fed a commercially available high-fat, moderate-protein, low-carbohydrate RMBD (n = 11, CAD diagnosed n = 8, healthy n = 3). Those in the second cohort were fed a commercially available moderate-fat, moderate-protein, high-carbohydrate kibble diet (KD) (n = 9: CAD diagnosed n = 6, healthy n = 3). The diet intervention period lasted approximately 4.5 months (median 135 days). Statistical analyses of the serum profiles across all dogs (n = 20) and the urine profiles of the CAD-diagnosed subset (n = 8) were performed. Results and Discussion: The KD cohort was found to have higher concentrations of methionine than the RMBD cohort, both in serum (all dogs, p < 0.0001) and in urine (CAD-only cohort, p < 0.0002), as well as cystathionine and 4-pyridoxic acid. Methionine plays important roles in homocysteine metabolism, and elevated levels have been implicated in various pathologies. The CAD (n = 14) cohort dogs showed starker metabolic changes in response to diet regarding these pathways compared to the healthy (n = 6) cohort. However, there was no significant change in CAD severity as a result of either diet. Likely due to the higher meat content of the RMBD, higher concentrations of several carnitines and creatine were found in the RMBD cohort. Citrulline was found in higher concentrations in the KD cohort. Our findings provide insight into the relationship between diet and the serum and urine metabolite profiles of canines. They also suggest that neither diet significantly affected CAD severity.

Hyperresistinemia in Obese Female Dogs With Mammary Carcinoma in Benign-Mixed Tumors and Its Correlation With Tumor Aggressiveness and Survival.

Resistin is associated with metabolic, inflammatory, and neoplastic disorders, and is also considered a prognostic marker in human oncology. Canine mammary tumors have epidemiological, clinical, biological, and genetic characteristics similar to those of women and are proposed as a comparative study model. Here, we evaluate the serum levels of resistin in female dogs with or without mammary carcinoma in mixed tumors (CBMT) and its correlation with the proliferative potential of the tumor, obesity, and survival. Eighty dogs grouped according to the presence (50) or absence (30) of CBMT, reproductive status and body condition were assessed for weight, fat percentage, and canine body mass index. The characteristic of the proliferative potential of the tumor (Ki-67) was evaluated. Ki-67 levels (p = 0.024), staging (p = 0.004), and grade (p = 0.016) influenced the survival of the female dogs. Through a multifactorial analysis, it could be seen that the parameters proliferation index (Ki-67) (p = 0.044) and staging (p = 0.036) influenced the survival of the animals. Neutered and overweight dogs from the control and CBMT groups showed hyperresistinemia. Ki-67 expression and resistin levels in dogs with CBMT were higher in overweight dogs than in dogs with normal weight (p = 0.0001). The survival rate of dogs with CBMT, obese and with high levels of resistin (8,400 µg L-1) was lower when compared to those with lower levels of resistin. These results showed an important relationship between hyperresistinemia, tumor proliferative potential and excessive body fat, suggesting that resistin levels may act as an interesting prognostic marker in patients with CBMT.

Mathematical Modelling Using Predictive Biomarkers for the Outcome of Canine Leishmaniasis upon Chemotherapy.

Prediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient's prognosis.

Vincristine and ivermectin combination chemotherapy in dogs with natural transmissible venereal tumor of different cyto-morphological patterns: A prospective outcome evaluation.

Vincristine is the first-line drug for the chemotherapy of canine transmissible venereal tumor (CTVT). Drug resistance is related to tumor cyto-morphological patterns of CTVT. There are anti-cancer properties of ivermectin, thus, a combination of ivermectin and vincristine could be an effective chemo-therapeutic treatment regimen for CTVT. Study aims, therefore, were to (1) assess the frequency of CTVT cyto-morphologies, and (2) evaluate treatment efficacy and possible adverse reactions to vincristine compared with a combination vincristine and ivermectin. Dogs (n = 41) with CTVT were characterized by tumor cyto-morphology and disease severity and of those, 20 were randomly allocated into two groups. There was a control group (G-Vin; n = 10) in which there was treatment with vincristine; and an experimental group (G-Iv/Vin; n = 10) in which there was treatment with the ivermectin/vincristine combination. Although dogs in the G-Iv/Vin group had more severe disease at the beginning of the study (P = 0.0031), the number of weeks and chemotherapy sessions until tumor remission were similar among dogs of the two groups, indicating both treatments were effective. There was a decrease in the leukocyte counts (P = 0.0020), related to neutropenia (P = 0.0371) in the G-Vin but not the G-Iv/Vin treatment group. There was no tumor resistance that developed during the study regardless of the treatment regimen used or tumor cytomorphology. In summary, the use of the vincristine/ivermectin combination was well tolerated and efficacious for CTVT treatment.

Immunogenicity and protection induced by recombinant Toxocara canis proteins in a murine model of toxocariasis.

Toxocariasis, a natural helminth infection of dogs and cats caused by Toxocara canis and T. cati, respectively, that are transmitted to mammals, including humans. Infection control is based currently on periodic antihelmintic treatment and there is a need for the development of vaccines to prevent this infection. MATERIALS AND METHODS: Eight potential vaccine candidate T. canis recombinant proteins were identified by in silico (rTcGPRs, rTcCad, rTcVcan, rTcCyst) and larval proteomics (rTES26, rTES32, rMUC-3 and rCTL-4) analyses. Immunogenicity and protection against infectious challenge for seven of these antigens were determined in a murine model of toxocariasis. C57BL/6 female mice were immunized with each of or combinations of recombinant antigens prior to challenge with 500 T. canis embryonated eggs. Levels of specific antibodies (IgG, IgG1, IgG2a and IgE) in sera and cytokines (IL-5, INF-É£ and IL-10) produced by antigens-stimulated splenocytes, were measured. Presence of specific antibodies to the molecules was measured in sera of T. canis-seropositive dogs and humans. RESULTS: All seven molecules were immunogenic in immunized mice; all stimulated significantly elevated levels of specific IgG, IgG1 or IgG2a and six were associated with elevated levels of specific IgE; all induced elevated production of IFN- É£ and IL-10 by splenocytes, but only the in silico-identified membrane-associated recombinants (rTcCad, rTcVcan, and rTcCyst) induced significantly increased IL-5 production. Vaccination with two of the latter (rTcCad and rTcVcan) reduced larval loads in the T. canis challenged mice by 54.3% and 53.9% (P < 0.0001), respectively, compared to unimmunized controls. All seven recombinants were recognized by T. canis-seropositive dog and human sera. CONCLUSION: The identification of vaccine targets by in silico analysis was an effective strategy to identify immunogenic T. canis proteins capable of reducing larval burdens following challenge with the parasite. Two recombinant proteins, rTcCad and rTcVcan, were identified as promising vaccine candidates for canine toxocariasis.