Influence of Various Factors on Circulating 25(OH) Vitamin D Concentrations in Dogs with Cancer and Healthy Dogs.

BACKGROUND: Low blood 25-hydroxyvitamin D (25(OH)D) concentrations have been associated with cancer in dogs. Little research has examined what other factors may affect 25(OH)D concentrations. OBJECTIVES: (1) To determine whether the presence of cancer (lymphoma, osteosarcoma, or mast cell tumor [MCT]) in dogs is associated with plasma 25(OH)D concentrations and (2) identify other factors related to plasma 25(OH)D concentrations in dogs. ANIMALS: Dogs newly diagnosed with osteosarcoma (n = 21), lymphoma (n = 27), and MCT (n = 21) presented to a tertiary referral oncology center, and healthy, client-owned dogs (n = 23). METHODS: An observational study design was used. Dietary vitamin D intake, sex, age, body condition score (BCS), muscle condition score (MCS), and plasma concentrations of 25(OH)D, 24,25-dihydroxyvitamin D (24,25(OH)2 D) (a marker of CYP24A1 activity), as well as ionized calcium (ICa), parathyroid hormone, and parathyroid hormone-related protein concentrations were measured. An analysis of covariance was used to model plasma 25(OH)D concentrations. RESULTS: Cancer type (P = 0.004), plasma 24,25(OH)2 D concentrations (P < 0.001), and plasma ICa concentrations (P = 0.047) had significant effects on plasma 25(OH)D concentrations. Effects of age, sex, body weight, BCS, MCS, and plasma PTH concentrations were not identified. A significant interaction between ICa and cancer was found (P = 0.005). Plasma 25(OH)D concentrations increased as ICa concentrations increased in dogs with cancer, whereas plasma 25(OH)D concentrations decreased as ICa concentrations increased in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Results support a relationship between cancer and altered vitamin D metabolism in dogs, mediated by plasma ICa concentrations. The CYP24A1 activity and plasma ICa should be measured in studies examining plasma 25(OH)D concentrations in dogs.

Evaluation of Weight Change During Carboplatin Therapy in Dogs With Appendicular Osteosarcoma.

BACKGROUND: The prevalence of cancer cachexia in veterinary medicine has not been studied widely, and as of yet, no definitive diagnostic criteria effectively assess this syndrome in veterinary patients. OBJECTIVES: (1) To determine the patterns of weight change in dogs with appendicular osteosarcoma treated with amputation and single-agent carboplatin during the course of adjuvant chemotherapy; and (2) to determine whether postoperative weight change is a negative prognostic indicator for survival time in dogs with osteosarcoma. ANIMALS: Eighty-eight dogs diagnosed with appendicular osteosarcoma. Animals were accrued from 3 veterinary teaching hospitals. METHODS: Retrospective, multi-institutional study. Dogs diagnosed with appendicular osteosarcoma and treated with limb amputation followed by a minimum of 4 doses of single-agent carboplatin were included. Data analyzed in each patient included signalment, tumor site, preoperative serum alkaline phosphatase activity (ALP), and body weight (kg) at each carboplatin treatment. RESULTS: A slight increase in weight occurred over the course of chemotherapy, but this change was not statistically significant. Weight change did not have a significant effect on survival. Institution, patient sex, and serum ALP activity did not have a significant effect on survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Weight change was not a prognostic factor in these dogs, and weight loss alone may not be a suitable method of determining cancer cachexia in dogs with appendicular osteosarcoma.

Owners' attitudes and practices regarding nutrition of dogs diagnosed with cancer presenting at a referral oncology service in Ontario, Canada.

OBJECTIVES: To investigate owner attitudes and dietary practices following cancer diagnosis in a dog. METHODS: A retrospective cross-sectional survey of 75 dog owners presenting with their dogs to a tertiary referral oncology service through a demographic questionnaire and in-person or telephone interviews regarding the dog's nutrition. RESULTS: Conventional diets (71%) were most commonly fed as a single diet to canine cancer patients followed by homemade cooked (7%) and homemade raw (4%). Several owners (18%) provided combinations of these diets. Owners reported some distrust towards conventional diets (51%). Appetite loss occurred in 35% of dogs and diet changes reported for 25% of dogs in the study involved exclusion of a conventional (63%) and/or inclusion of a homemade (54%) component. 90% of owners noted the diet change was associated with the cancer diagnosis. Supplements were given by 39% of owners. 85% of owners highly valued veterinary nutritional advice. CLINICAL RELEVANCE: Following a cancer diagnosis, dog owners appear to change their approach to managing their dog's nutrition. Given the value owners place on veterinary nutritional advice, veterinarians have a key role in guiding nutritional management of the canine cancer patient.

Comparison of concurrent imaging modalities for staging of dogs with appendicular primary bone tumours.

This study assessed the use of whole body computed tomography (CT) for the evaluation of metastasis in dogs with primary appendicular bone tumours compared to long bone survey radiography, bone scintigraphy and thoracic radiographs. Fifteen dogs were included in this pilot study. A construct reference standard was used for detection of bone metastasis, and negative thoracic radiographs were compared against CT. Definitive lesions were only identified on bone scintigraphy. Not all lesions agreed with the construct reference standard. No definitive lesions were identified on survey radiographs or CT. Lesions were identified on thoracic CT that were not visible radiographically. Equivocal ground glass pulmonary lesions progressed in three of four cases. Whole body CT was not a suitable alternative to bone scintigraphy; however, it was useful as an adjunctive diagnostic modality. Pulmonary lesions were visible on CT that were not seen radiographically and ground glass pulmonary lesions in dogs should be considered suspicious for metastasis.

The immunophenotype of peripheral blood lymphocytes in clinically healthy dogs and dogs with lymphoma in remission.

Lymphoma is a common cancer of dogs that frequently is treated with chemotherapy or radiation therapy. Response to therapy is variable and currently available diagnostic tests do not reliably predict response to therapy. Treatment for lymphoma often results in lymphopenia, but it is unknown whether the changes in circulating lymphocytes result from generalized or specific reduction of lymphocytes. In this study, blood lymphocytes from 12 clinically healthy dogs, 10 dogs in remission because of treatment for B-cell lymphoma, and 8 dogs in remission from T-cell lymphoma were analyzed by flow cytometry by using a panel of 20 antibodies reactive with canine leukocyte antigens. Results identified similar lymphocyte parameters in treated dogs regardless of the type of lymphoma. Treated dogs had >50% reduction in blood lymphocyte concentration, and an absolute decrease in most subsets of lymphocytes. Both groups of treated dogs had relative increases in the proportion of CD3+, T-cell receptor (TCR)alphabeta+, and CD90+ lymphocytes, and a decreased proportion of CD45RA+ cells. In addition, dogs with T-cell lymphoma in remission had a significant increase in the proportion of CD49d+ lymphocytes. These findings were interpreted as representing likely suppression of lymphocyte regeneration by chemotherapy, with a relative increase in the proportion of memory over naive lymphocytes. Lack of correlation with the T- or B-cell origin of the initial lymphoma suggested that, by using flow cytometric methods, residual circulating neoplastic cells could not be detected. However, the changes in the lymphocyte profile of dogs treated with chemotherapy may have relevance to their immunocompetence.

Flow cytometric immunophenotype of canine lymph node aspirates.

Increasing availability of reagents able to distinguish subtypes of lymphocytes and other leukocytes has enabled greater understanding of lymphocyte biology and pathology in the dog. Lymphocytes in circulation most commonly are subjected to immunophenotypic assessment by flow cytometry, but needle aspirates of lymph nodes can be similarly suitable for immunophenotypic examination. In this investigation, the feasibility of immunophenotyping samples obtained by needle aspiration of lymph nodes from 32 dogs with no physical abnormalities and 6 dogs with lymphoma was determined. In addition, samples from 6 dogs were stored overnight at 4 degrees C and reanalyzed 24 hours later. For each sample, stained smear preparations were examined microscopically for lymphocyte morphology, neoplasia, and the presence of inflammatory cells. Expression of antigens on a corresponding sample of aspirated cells was determined by flow cytometric detection of antibody binding on a minimum of 10,000 events. The distribution of data was determined with Anderson-Darling tests, and reference intervals incorporating the central 95% of values were established. Adequate samples were obtained from 30 of 32 clinically normal dogs. Immunophenotypic results after 24 hours of storage were consistent with those obtained immediately after sampling. Reference intervals for lymphocyte subsets from normal dog lymph nodes were similar to the proportions of CD3+, CD4+, CD8+, and CD21+ lymphocytes found in blood. Aspirates of enlarged lymph nodes from dogs with lymphoma were readily classified by this technique. Aspiration of lymph nodes from dogs for comprehensive analysis by flow cytometry is feasible and applicable to immunophenotyping of lymphoma.

Myasthenia gravis and masticatory muscle myositis in a dog.

A 21-month-old, castrated male Vizsla was presented for pelvic limb weakness, difficulty opening his mouth, ptyalism, voice change, and urinary incontinence. Myasthenia gravis and masticatory myositis were diagnosed. The unusual clinical findings, diagnosis, treatment, and case outcome are described, followed by a brief discussion of myasthenia gravis and masticatory myositis.

Potential role for extracellular glutathione-dependent ferric reductase in utilization of environmental and host ferric compounds by Histoplasma capsulatum.

The mammalian host specifically limits iron during Histoplasma capsulatum infection, and fungal acquisition of iron is essential for productive infection. H. capsulatum expresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. The H. capsulatum siderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).

Assessment of lithium dilution cardiac output as a technique for measurement of cardiac output in dogs.

OBJECTIVES: To determine agreement of cardiac output measured by use of lithium dilution cardiac output (LiDCO) and thermodilution cardiac output (TDCO) techniques in dogs and to determine agreement of low- and high-dose LiDCO with TDCO. ANIMALS: 10 dogs (7 males, 3 females). PROCEDURE: Cardiac output was measured in anesthetized dogs by use of LiDCO and TDCO techniques. Four rates of cardiac output were induced by occlusion of the caudal vena cava, changes in depth of anesthesia, or administration of dobutamine. Lithium dilution cardiac output was performed, using 2 doses of lithium chloride (low and high dose). Each rate of cardiac output allowed 4 comparisons between LiDCO and TDCO. RESULTS: 160 comparisons were determined of which 68 were excluded. The remaining 92 comparisons had values ranging from 1.10 to 12.80 L/min. Intraclass correlation coefficient (ICC) between low-dose LiDCO and TDCO was 0.9898 and between high-dose LiDCO and TDCO was 0.9896. When all LiDCO determinations were pooled, ICC was 0.9894. For determinations of cardiac output < 5.0 L/min, ICC was 0.9730. Mean +/- SD of the differences of TDCO minus LiDCO for all measurements was -0.084+/-0.465 L/min, and mean of TDCO minus LiDCO for cardiac outputs < 5.0 L/min was -0.002+/-0.245 L/min. CONCLUSIONS AND CLINICAL RELEVANCE: The LiDCO technique is a suitable substitute for TDCO to measure cardiac output in dogs. Use of LiDCO eliminates the need for catheterization of a pulmonary artery and could increase use of cardiac output monitoring, which may improve management of cardiovascularly unstable animals.

Pathogenesis of Histoplasma capsulatum.

Histoplasma capsulatum is well adapted to be infectious and pathogenic for humans. As a soil fungus with no known requirement for interacting with a mammalian host as part of an obligate lifecycle, its plethora of strategies for successful pathogenesis is particularly remarkable. These features include the dimorphic mold-yeast transition, entry into host macrophages, subcellular localization, intracellular survival and proliferation during active infection, and persistence during clinically inapparent infection with the capacity for reactivation. To thrive within the harsh environment of a professionally phagocytic and antimicrobial host cell, H. capsulatum displays mechanisms for modulating its microenvironmental pH level, resisting host reactive oxygen and nitrogen intermediates and degradative enzymes, and withstanding nutrient starvation conditions, including acquisition of iron and calcium and biosynthesis of nucleic acid precursors. Attention has been focused on identifying virulence-associated phenotypic traits and genes that are differentially expressed under relevant conditions, such as yeast morphotype-specific genes and genes that are up-regulated during infection. These studies, together with the increasing ability to perform molecular genetic manipulations in this fungus, may yield novel antifungal drug or vaccine targets as well as elucidating pathogenic mechanisms.

Quantification of hepatic and renal cortical echogenicity in clinically normal cats.

OBJECTIVE: To quantitatively determine echogenicity of the liver and renal cortex in clinically normal cats. ANIMALS: 17 clinically normal adult cats. PROCEDURE: 3 ultrasonographic images of the liver and the right kidney were digitized from video output from each cat. Without changing the ultrasound machine settings, an image of a tissue-equivalent phantom was digitized. Biopsy specimens of the right renal cortex and liver were obtained for histologic examination. Mean pixel intensities within the region of interest (ROI) on hepatic, renal cortical, and tissue-equivalent phantom ultrasonographic images were determined by histogram analysis. From ultrasonographic images, mean pixel intensities for hepatic and renal cortical ROI were standardized by dividing each mean value by the mean pixel intensity from the tissue-equivalent phantom. RESULTS: The mean (+/- SD) standardized hepatic echogenicity value was 1.06 +/- 0.02 (95% confidence interval, 1.02 to 1.10). The mean standardized right renal cortical echogenicity value was 1.04 +/- 0.02 (95% confidence interval, 1.01 to 1.08). The mean combined standardized hepatic and renal cortical echogenicity value was 1.02 +/- 0.05 (95% confidence interval, 0.99 to 1.04). CONCLUSIONS AND CLINICAL RELEVANCE: Quantitative determination of hepatic and renal cortical echogenicity in cats is feasible, using histogram analysis, and may be useful for early detection of diffuse parenchymal disease and for serially evaluating disease progression.

Cats surviving natural infection with Cytauxzoon felis: 18 cases (1997-1998).

Eighteen cats surviving natural infection with Cytauxzoon felis were identified. All cats came from a limited geographic area in northwestern Arkansas and northeastern Oklahoma. Clinical signs in most cats were similar to those described for cytauxzoonosis; however, 4 cats were asymptomatic. All cases were initially diagnosed by microscopic identification of signet ring-shaped piroplasms in erythrocytes of peripheral blood smears. Four of 4 cats tested had detectable serum antibodies to C felis. Four different cats were positive by polymerase chain reaction (PCR). Partial sequencing of the PCR product from 1 cat revealed >99% homology with the reported sequence of C felis. Repeated examination of blood smears from 12 cats revealed that the erythroparasitemia was generally persistent for the duration of follow-up (3-154 days). Survival did not seem dependent on treatment, as only 1 cat was treated with a drug with potential antiprotozoal activity (imidocarb dipropionate), and 4 cats received no treatment. The findings of this study may indicate the existence of a less virulent strain of C felis.

Determination of beta-glucosidase enzymatic function of the histoplasma capsulatum H antigen using a native expression system.

The Histoplasma capsulatum H antigen is a major secreted glycoprotein of this pathogenic fungus that is a target of humoral and cell-mediated host responses. Its predicted protein sequence displays homology to beta-glucosidases of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of the protein carrying a carboxyl-terminus oligohistidine tag in the native fungal background to facilitate proper glycosylation and folding of a product that could then be purified from culture supernatants using nickel affinity chromatography. The recombinant protein was expressed and secreted by a transformant carrying the modified gene under the control of its native promoter. The purified protein from the native expression system showed beta-glucosidase enzymatic activity in substrate gels and quantitative microplate assays. This activity was blocked by glucosidase-specific inhibitors. These results are the first direct demonstration of the function of this protein, and show the utility of expression in a native system to achieve post-translational modification necessary for structural and functional integrity.

Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay.

We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages. The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts. Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells. Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake.

The effects of a unilateral ultrasound-guided renal biopsy on renal function in healthy sedated cats.

Complications of renal biopsies are well documented except for the change in renal function after a biopsy. Eighteen healthy, adult cats were divided into two groups (n = 9 cats/group). For the measurement of global and split renal function, Group 1 used the renal uptake of 99mTc-DTPA and Group 2 used the renal uptake of 99mTc-MAG3. Scintigraphic data were collected on days (-4), (-3), 0, 1, 2, and 4 post renal biopsy. Using ultrasound guidance, biopsies were taken from the right renal cortex on dO, before acquiring scintigraphic images. P - values less than 0.10 were considered significant due to the limited number of observations. The only statistically significant change (p = 0.08) in global renal function detected was by day following a unilateral renal biopsy. Cats imaged using 99mTc-MAG3 had discernible liver activity. A unilateral, ultrasound guided renal biopsy has minimal effect on renal function in normal, healthy sedated cats.

Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum.

Understanding how pathogens survive within the host cell is of paramount importance in the development of vaccines and therapeutic agents. This task has been particularly daunting in the study of fungal pathogens due to the lack of easily manipulated genetic systems. In recent years several molecular genetic reporter systems have been developed to identify genes expressed during the infection process and potential virulence determinants. The development of one method in particular, in vivo expression technology (IVET), has led to the discovery of several genes from various bacterial pathogens necessary for survival during infection. The recent development of molecular genetic tools for Histoplasma capsulatum has enabled us to adapt the IVET technology for this pathogenic fungus utilizing the URA5 gene, which is essential for H. capsulatum survival in mice and in cultured macrophages, as a reporter of in vivo gene expression. We report the first successful application of IVET screening of a fungal pathogen for genes expressed exclusively during infection.

Prevalence of American trypanosomiasis (Chagas disease) among dogs in Oklahoma.

OBJECTIVE: To determine the prevalence of Trypanosoma cruzi infection among dogs in Oklahoma. DESIGN: Cross-sectional study. ANIMALS: 301 owned or impounded dogs related by ownership or general geographic location to 3 dogs determined to have trypanosomiasis. PROCEDURES: Blood samples were obtained from dogs between November 1996 and September 1997. Infection status was determined by use of a radioimmunoprecipitation assay. Second blood samples were obtained from some of the seropositive dogs for study by hemoculture and polymerase chain reaction (PCR) assay. Sites where infected dogs were found were inspected for triatomine insects, and light traps were used for vector trapping. RESULTS: 11(3.6%) dogs were seropositive for T. cruzi infection. Ten of the 11 were owned rural hunting dogs. Protozoal organisms isolated from the blood of 1 seropositive dog were identified as T. cruzi by PCR testing. Only 1 adult Triatoma sanguisuga was captured in a light trap at a site near infected dogs; this insect was not infected. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that T. cruzi is enzootic in eastern Oklahoma. Measures that would reduce the risk of dogs acquiring T. cruzi infection are unlikely to be acceptable to their owners, and no effective drugs are available for treatment. The presence of T. cruzi-infected dogs poses a threat of transmission to persons at risk of exposure to contaminated blood Veterinarians who practice in the southern United States should be cognizant of this blood borne zoonosis and educate all personnel about appropriate precautions.