Veronica Marchetti

Analytical performances of d-ROMs test and BAP test in canine plasma. Definition of the normal range in healthy Labrador dogs.

An high level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress, an emerging health risk factor involved in the aging and in many diseases, including inflammatory, infectious and degenerative disorders, either in humans or in animals. In the last years some assays panels have been developed to globally evaluate the oxidative balance by means of the concomitant assessment of ROS production and antioxidant system capability. In this report, the validation trials of d-ROMs (Reactive Oxygen Metabolites— derived compounds) and BAP (Biological Antioxidant Potential) tests in canine specie are described and also the specific referral ranges are calculated in a Labrador population. The results of linearity, precision and accuracy trials show that both tests exhibit good to excellent analytical performances. The possibility of measuring oxidative stress in vivo with simple, cheap and accurate tests, d-ROMs test and BAP test, provides for the veterinarians a very suitable tool to monitor oxidative stress and to correctly choice of eventual antioxidant supplementations in diseases proven related to oxidative stress in animals and particularly in dogs. Further studies will be useful to confirm this possibility.

First-line metronomic chemotherapy in a metastatic model of spontaneous canine tumours: a pilot study

Metronomic chemotherapy—the low-dose, long term and frequently administered chemotherapy—has revealed in these years an important impact on the stabilization of cancer disease for its known antiangiogenic effects, prolonged clinical benefits and the improved quality of life of several cancer patients, without any high grade toxicity [1–3]. Both the low cost and the oral administration of the drugs are key characteristics of this schedule and may offer important social advantages [4]. Anecdotical case reports [5–7] and experiences in small subsets of patients enrolled in retrospective clinical studies [8–10] on metastatic cancers have been recently published about the use of metronomic therapy as a first-line treatment. These point out the possible importance of metronomic chemotherapy as an alternative approach to first-line therapy in frail patients requiring palliation or patients refusing the standard chemotherapy for its impact on the quality of life. However, no data of prospective clinical trials on first line metronomic chemotherapy are currently available in metastatic cancer human patients. The veterinary medical oncology has advanced dramatically over the past few decades, because of the successful application of a number of conventional chemotherapeutic drugs to the cancer conditions diagnosed in veterinary patients [11]. Veterinary oncology cases often present a unique opportunity to investigate novel drugs and treatment schedules providing many in vivo information to the larger medical community and giving new effective options for dogs themselves [12]. As well recently pointed out by Paoloni and Khanna [13], these studies may also have a great translational relevance, predicting new therapies and related surrogate markers in human beings because pet dogs with cancers might assist the transition between mouse models and human patients. Moreover, in the clinical practice, veterinarians and their clients are generally less willing to accept a high degree of side effects, which most often results in lower drug doses than the ones that are used in human oncology. The aim of the present pilot study was to test a first-line metronomic oral combination of cyclophosphamide (CTX) and celecoxib (CXB) in canine metastatic spontaneous tumours, characterizing possible biomarkers to translate in human clinical research.

Hepatozoonosis in a dog with skeletal involvement and meningoencephalomyelitis

A 15-month-old, female mongrel dog was presented with a 6-week history of inappetence, weight loss, and tetraparesis. Physical examination revealed weakness, poor body condition, mild fever, pale mucous membranes, and diffuse muscle atrophy. The right hind limb was painful and edematous, with large ecchymoses. The femur was irregular on palpation and moderate popliteal lymphadenopathy was evident. Results of a CBC showed severe anemia with mild regeneration, an inflammatory leukogram with 90% of neutrophils parasitized by Hepatozoon sp. gamonts, and moderate thrombocytopenia. A bone marrow aspirate had myeloid hyperplasia and contained a few extracellular Hepatozoon meronts and a few intracellular gamonts within neutrophils. Serum chemistry abnormalities included hypoalbuminemia, hyperglobulinemia, hypoglycemia, hypercalcemia, hyperphosphatemia, and elevated alkaline phosphatase activity. Radiologic findings of the right femur included periosteal bone proliferation and lesions compatible with osteomyelitis. A fine needle aspirate specimen from the bone lesion had neutrophilic inflammation; 36% of the neutrophils contained Hepatozoon gamonts. Results of cerebrospinal fluid analysis included a protein concentration of 37 mg/dL and marked mononuclear pleocytosis (243 cell/microL) with a predominance of lymphocytes. An ELISA was positive for Hepatozoon canis and PCR results with DNA sequencing confirmed infection with this organism. A diagnosis of hepatozoonosis with skeletal involvement and meningoencephalomyelitis was made. The dog recovered almost completely neurologically and had no gamonts in the blood after 60 days of therapy with imidocarb dipropionate and prednisone. This is an unusual case of canine hepatozoonosis involving neurologic signs and a periosteal reaction more typical of H. americanum infection and rarely reported in dogs infected with H. canis.

Evaluation of fungicidal efficacy of benzalkonium chloride (Steramina G u.V.) and Virkon-S against Microsporum canis for environmental disinfection

The aim of this study is to evaluate the antifungal efficacy of Steramina G u.v. (10% solution of alkyldimetylbenzylammonium chloride; Formenti Grünenthal) and Virkon-S (multipurpose system; Antec International) against Microsporum canis-infected hairs and spores. Samples were collected from a random sample of household cats and from subjects from catteries. Seventy M. canis-positive hairbrushes containing furs, keratin scales and other organic material were treated with each of the two disinfectants, using concentrations recommended by the manufacturers instructions (2% and 1% for Steramina G u.v. and Virkon-S, respectively). Each brush remained in contact with the antifungal solution for 10 min. After this period, the brushes were air-dried, then seeded into mycobiotic agar, and incubated for up to 21 days at 28°C. The disinfectants were considered effective if dermatophytes failed to grow. Steramina G u.v. was effective in 97.14% of samples and Virkon-S in 87.14%. The antifungal activity of Steramina G u.v. against M. canis was significantly higher (p < 0.05) than that of Virkon-S.

Redox status evaluation in dogs affected by mast cell tumour.

Oxidative stress status has been evaluated in depth in human medicine and its role in carcinogenesis has been clearly established. The purpose of this prospective study was to evaluate antioxidant concentrations and oxidative stress in dogs with mast cell tumours (MCTs) that had received no previous treatments, and to compare them to healthy controls. In 23 dogs with mast cell tumour and 10 healthy controls, oxidative status was assessed using the Reactive Oxygen Metabolites-derived compounds (d-ROMs) test, antioxidant activity was measured by the Biological Antioxidant Potential (BAP) test, and α-tocopherol levels were evaluated using high-performance liquid chromatography and ultraviolet analysis. At baseline, dogs with MCT had significantly higher d-ROMs (P < 0.00001) and lower BAP (P < 0.0002) compared with healthy controls. However, no significant difference was observed for α-tocopherol (P = 0.95). Results suggest that oxidative stress pattern and oxidative defence barrier are altered in dogs with newly diagnosed MCT compared with control dogs. Future studies are needed in order to assess the prognostic role of oxidative stress and to evaluate the impact of different therapeutic approaches.

Morphometrical approach for predicting regional lymph node micrometastatic load in canine mast cell tumours: preliminary results.

Canine cutaneous mast cell tumours (MCTs) have a variable biologic behaviour, and accurate staging is necessary to dictate therapy and predict outcome. Regional lymph node (RLN) involvement is a relevant prognostic factor. While obvious lymph node (LN) metastases are relatively easy to be diagnosed, micrometastatic disease recognition is challenging. The main aim of the study was to evaluate the number of mast cells (MCs) in the LNs of clinically healthy dogs (n = 4, group 1), dogs with inflammatory diseases (n = 31, group 2) and dogs with cutaneous MCT (n = 27, group 3), including animals with no RLN metastases (subgroup 3.1), those with occasional MCs in RLNs (3.2) and those with obvious RLN metastasis (3.3). MCs also were morphometrically evaluated for the following nuclear parameters: mean nuclear area (MNA), mean nuclear perimeter (MNP), largest to smallest diameter length (LS ratio), mean nuclear form factor and coefficient of variation of nuclear area. The average percentages of MCs were 0.0 and 0.01 in groups 1 and 2, respectively, and 0.07, 2.4 and 47.1 in subgroup 3.1, 3.2 and 3.3. MNA and MNP were significantly higher in subgroup 3.3 than in group 2 (P < 0.05). MNA and MNP in subgroup 3.2 suggested the presence of neoplastic MCs; this prediction of micrometastatic load correlated with outcome. Analysis of preliminary results shows that nuclear morphometry is useful to detect micrometastatic disease in RLN of dogs bearing cutaneous MCTs.

Prognostic role of the product of serum calcium and phosphorus concentrations in dogs with chronic kidney disease: 31 cases (2008–2010)

OBJECTIVE To investigate serum calcium-phosphorus concentration product (sCaPP) as a predictor of mortality rate in dogs with chronic kidney disease (CKD). DESIGN Retrospective case-control study. ANIMALS 31 dogs with definitive CKD and 35 apparently healthy dogs. PROCEDURES All dogs had been referred for nephrological consultation between December 2008 and December 2010. Dogs with CKD had stable disease for ≥ 3 months. On the basis of glomerular filtration rate < 60 mL/min/m(2), 13 of the 35 apparently healthy dogs were subsequently classified as having early CKD. Disease stage among dogs was determined on the basis of plasma creatinine concentration as follows: stage 1, < 123.7 μmol/L (n = 13), stage 2, 123.7 to 176.8 μmol/L (7); stage 3, 185.6 to 442 μmol/L (13); or stage 4, > 442 μmol/L (11). For each dog, serum concentrations of ionized and total calcium and phosphorus were evaluated once; the latter 2 variables were used to determine sCaPP. RESULTS The sCaPP differed significantly between the 22 healthy dogs and dogs with stage 3 or stage 4 CKD. The proportion of dogs with sCaPP > 70 mg(2)/dL(2) increased with stage of disease. Mortality rate among the 24 dogs with sCaPP > 70 mg(2)/dL(2) was higher than that among the 42 dogs with sCaPP ≤ 70 mg(2)/dL(2). Dogs with sCaPP > 70 mg(2)/dL(2) had a comparatively lower survival rate, and risk of death was 4.2 times as high as risk for dogs with sCaPP ≤ 70 mg(2)/dL(2). CONCLUSIONS AND CLINICAL RELEVANCE For dogs with CKD, sCaPP > 70 mg(2)/dL(2) appeared to be a negative prognostic indicator, which was not influenced by the concomitant serum concentrations of phosphorus and total or ionized calcium.

Evaluation of erythrocytes, platelets, and serum iron profile in dogs with chronic enteropathy.

The aim of this study is to evaluate iron status, erythrocyte, and platelet modifications in dogs with chronic enteropathy (CE). Dogs were grouped as food-responsive diarrhea (FRD, n = 11), antibiotic-responsive diarrhea (ARD, n = 5), and steroid-responsive diarrhea (SRD, n = 6) relating to therapeutic-response. Clinical and haematological findings, evidence of gastrointestinal blood loss, and iron metabolism were evaluated before and after treatment. A mild normocytic or microcytic anemia and thrombocytosis were identified, respectively in 18.0% and 31.8% of CE dogs. No significant differences between pre- and posttreatment of hematocrit, haemoglobin, and mean corpuscular volume, platelet count and mean platelet volume were found. Statistical analysis pointed out significant differences between pre- and posttreatment in serum iron (P < .03) and unsaturated iron binding capacity (UIBC) (P < .01). No significant correlations were found between these parameters and canine Inflammatory Bowel Disease activity index and pattern of CE as well.

Pancreatic Islet Cell Tumor Secreting Insulin-Like Growth Factor Type-II in a Dog

7-year-old, intact female, Gordon Setter was examined for a 6-month history of progressive weakness and ataxia without loss of appetite or change in weight. The owner reported a slight improvement in the signs after food consumption. The dog was kept indoors, regularly vaccinated, and fed a commercial maintenance diet. Physical examination revealed weakness, difficulty in holding quadrupedal posture, and mild muscle hypotrophy. No abnormalities were detected in the CBC, morphologic evaluation of the smear, and coagulation profile. Biochemical profile showed moderate hypoglycemia on fasting (57 mg/dL; range 80–120 mg/dL). Abdominal ultrasound showed an hypoechoic pancreatic lesion of 23 mm in diameter, with indistinct margins and poorly contrasting with adjoining structures. Eco-guided fine needle biopsy of the lesion was performed. The cytologic specimen contained a large number of naked nuclei on a cytoplasmic background with indistinct margins, occasionally acinar structures with moderate anisokaryosis. The cytologic pattern, together with clinical signs, suggested neuroendocrine tumor. Abnormalities were not detected on chest X-ray in 3 standard projections. Serum concentration of insulin was 0.5 mIU/mL (range 4–16 mIU/mL). Serum concentration of insulin-like growth factor type II (IGF-II) was evaluated by immunoradiometric assay (IRMA) after chromatographic separation. Five hundred microliters of serum were obtained from the dog and gel-filtered by fast protein liquid chromatography on HyPrep Sephacryl S-200 High Resolution column (GE Healthcare, Amersham Place, Little Chalfont, Buckinghamshire, UK) in a buffer containing 50 mM NaH2PO4, 0.15 M NaCl, 0.02% NaN3 ,p H 7.2. Samples were eluted at 0.8 mL/min and collected at 3minute intervals. 1 The 44 fractions collected were pooled and tested for IGF-II immunoreactivity as follows: fractions 8–11 corresponding to the 150 kDa ternary complex, fractions 12–16 corresponding to the 45 kDa binary complex, and fractions 24–27 corresponding to the free form of IGF-II. The fraction of IGF-II bound to insulin-like growth factor binding protein (IGFBP)-3 and acid labile subunit (ALS) forming a 150-kDa complex was 0.8 ng/mL. Similarly to normal dogs, IGF-II was only measurable in the 150 kDa region and undetectable in the others. IGF-II was measured by IRMA with reagents kit provided by DSL, a on acid ethanol pretreated samples. 2 The sensitivity of the assay was 0.13 ng/mL; the intra-assay and interassay coefficients of variation were 5.3 and 8.7%, respectively. No detectable cross-reactivity was found against IGF-I, up to 480,000 ng/mL, proinsulin, up to 2mg/mL, and insulin, up to 4.3mg/mL.

Spectral waveform analysis of intranodal arterial blood flow in abnormally large superficial lymph nodes in dogs

OBJECTIVE To evaluate pulsed-wave Doppler spectral parameters as a method for distinguishing between neoplastic and inflammatory peripheral lymphadenopathy in dogs. SAMPLE POPULATION 40 superficial lymph nodes from 33 dogs with peripheral lymphadenopathy. PROCEDURES 3 Doppler spectral tracings were recorded from each node. Spectral Doppler analysis including assessment of the resistive index, peak systolic velocity-to-end diastolic velocity (S:D) ratio, diastolic notch velocity-to-peak systolic velocity (N:S) ratio, and end diastolic velocity-to-diastolic notch velocity ratio was performed for each tracing. Several calculation methods were used to determine the Doppler indices for each lymph node. After the ultrasonographic examination, fine needle aspirates or excisional biopsy specimens of the examined lymph nodes were obtained, and lymphadenopathy was classified as either inflammatory or neoplastic (lymphomatous or metastatic) via cytologic or histologic examination. Results of Doppler analysis were compared with cytologic or histopathologic findings. RESULTS The Doppler index with the highest diagnostic accuracy was the S:D ratio calculated from the first recorded tracing; a cutoff value of 3.22 yielded sensitivity of 91%, specificity of 100%, and negative predictive value of 89% for detection of neoplasia. Overall diagnostic accuracy was 95%. At a sensitivity of 100%, the most accurate index was the N:S ratio calculated from the first recorded tracing; a cutoff value of 0.45 yielded specificity of 67%, positive predictive value of 81%, and overall diagnostic accuracy of 86.5%. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that noninvasive Doppler spectral analysis may be useful in the diagnosis of neoplastic versus inflammatory peripheral lymphadenopathy in dogs.