Update on antifungal therapy.

Fungal pathogens are becoming increasingly important for human and small animal medicine. This article highlights many standards-of-care and new agents for treatment of these pathogens for small animals and people.     

Multiresidue assay for benzimidazole anthelmintics by liquid chromatography and confirmation by gas chromatography/selected-ion monitoring electron impact mass spectrometry

A multiresidue method utilizing all-disposable labware has been developed for 8 benzimidazole anthelmintics from ovine, bovine, and swine muscle and liver tissues. After an initial extraction with ethyl acetate and subsequent evaporation, a 3-component extraction using hexane, ethanol, and 0.2N HCl was used for final cleanup. Clean extracts were produced for separation and determination by reverse-phase liquid chromatography at 298 nm, using methanol and aqueous buffer as mobile phase. A synthesized internal standard, 2-(n-butylmercapto)benzimidazole, was used for quantitation of all drugs. Results are included along with statistical information verifying the performance of the method. Spiked control tissues and incurred drug tissues were used for an intralaboratory study with a concentration range of 50-1470 ppb. A series of standard curves at 0, 50, 100, and 200 ppb were analyzed. Overall recovery at the 100 ppb level averaged 92% (CV 8%) in liver tissues, across all 3 species and 88% (CV 5%) in muscle tissues across all 3 species. Results were confirmed by gas chromatography/mass spectrometry with acid hydrolysis of the remaining extract in 2N HCl followed by re-extraction of the amine and derivatization to the tert-butyldimethylsilyl derivative. The anthelmintics were identified by gas chromatography/selected ion monitoring electron-impact mass spectrometry. Ion ratio measurements were taken and compared to standard material. CVs averaged 10% or less for all drugs tested.     

Effects of Carbon Dioxide Exposure on Intensively Cultured Rainbow Trout Oncorhynchus mykiss: Physiological Responses and Fillet Attributes

Rainbow trout Oncorhynchus mykiss (261.6 × 24.7 g initial weight, mean × SEM) at 13.1 × 0.2 C were exposed for 94 d to one of three CO₂ treatments: control (22.1 × 2.8 mg/L), medium (34.5 × 3.8 mg/L), or high (48.7 × 4.4 mg/L). Trout were checked daily for survival, and fish were sampled at 0, 28, 56, and 84 d for physiological responses, growth, and fillet quality assessments. Trout were also challenged to a 15-min crowding stress at 93 d to assess their ability to initiate a stress response during hypercapnia. Chronically exposed trout showed nearly 100% survival through 84 d exposure (1 of 1,500 fish died). Growth and physiological results showed that increasing elevated CO₂, concentrations result in corresponding decreased growth rates and CO₂specific physiological parameters: The medium and high CO₂ treatments had significantly slower growth and subsequently smaller fish by 84 d. Exposed trout also showed significantly ( P < 0.05) decreased plasma chloride for medium and high CO₂ treatments compared to the control from 28 through 84 d. Decreased growth and smaller fish in the medium and high CO₂ treatments resulted in correspondingly smaller fresh and smoked fillet weights. Chronic CO₂ exposure did not result in notable changes in ultimate muscle pH. Exposure to 15-min crowding stress at 93 d resulted in significant changes in hematocrit, plasma cortisoI, glucose, and chloride for all treatment groups. CO₂-specific changes were detected in hematocrit, plasma cortisoI, and plasma chloride responses following the 15-min crowding stress.     

Estimation of the relative bioavailability of copper sources in chicks fed on conventional dietary amounts

1. Tissue accumulation of Cu from dietary additions of 0, 5, 10, or 20 mg/kg Cu as reagent grade Cu acetate and feed grade Cu carbonate was determined in day‐old chicks fed on conventional maize‐soyabean meal starter diets (5.41 mg/kg Cu as‐fed basis) for 3 weeks.

2. Average daily food intake, daily weight gain and food conversion were similar among treatments.

3. There were linear increases in plasma and liver Cu concentrations (P< 0.01) as dietary Cu increased.

4. Bioavailability of Cu as carbonate was 0.66 that of Cu in the acetate based on the multiple regression slope ratio of liver Cu concentration on added dietary Cu. Although responses for the two Cu sources did not differ significantly, the relative bioavailability of the Cu carbonate was similar (0.66 vs 0.68) to that obtained in an earlier study (Ledoux et al., 1991) with greater dietary Cu contents (150, 300 and 450 mg/kg) in which the slopes of the equations representing the two sources differed (P<0.05).     

Proposed criteria for classification of acute myeloid leukemia in dogs and cats

Blood and bone marrow smears from 49 dogs and cats, believed to have myeloproliferative disorders (MPD), were examined by a panel of 10 clinical pathologists to develop proposals for classification of acute myeloid leukemia (AML) in these species. French-American-British (FAB) group and National Cancer Institute (NCI) workshop definitions and criteria developed for classification of AML in humans were adapted. Major modifications entailed revision of definitions of blast cells as applied to the dog and cat, broadening the scope of leukemia classification, and making provisions for differentiating erythremic myelosis and undifferentiated MPD. A consensus cytomorphologic diagnosis was reached in 39 (79.6%) cases comprising 26 of AML, 10 of myelodysplastic syndrome (MDS), and 3 of acute lymphoblastic leukemia (ALL). Diagnostic concordance for these diseases varied from 60 to 81% (mean 73.3 +/- 7.1%) and interobserver agreement ranged from 51.3 to 84.6% (mean 73.1 +/- 9.3%). Various subtypes of AML identified included Ml, M2, M4, M5a, M5b, and M6. Acute undifferentiated leukemia (AUL) was recognized as a specific entity. M3 was not encountered, but this subclass was retained as a diagnostic possibility. The designations M6Er and MDS-Er were introduced where the suffix "Er" indicated preponderance of erythroid component. Chief hematologic abnormalities included circulating blast cells in 98% of the cases, with 36.7% cases having >30% blast cells, and thrombocytopenia and anemia in approximately 86 to 88% of the cases. Bone marrow examination revealed panmyeloid dysplastic changes, particularly variable numbers of megaloblastoid rubriblasts and rubricytes in all AML subtypes and increased numbers of eosinophils in MDS. Cytochemical patterns of neutrophilic markers were evident in most cases of Ml and M2, while monocytic markers were primarily seen in M5a and M5b cases. It is proposed that well-prepared, Romanowsky-stained blood and bone marrow smears should be examined to determine blast cell types and percentages for cytomorphologic diagnosis of AML. Carefully selected areas of stained films presenting adequate cellular details should be used to count a minimum of 200 cells. In cases with borderline diagnosis, at least 500 cells should be counted. The identity of blast cells should be ascertained using appropriate cytochemical markers of neutrophilic, monocytic, and megakaryocytic differentiation. A blast cell count of > 30% in blood and/or bone marrow indicates AML or AUL, while a count of < 30% blasts in bone marrow suggests MDS, chronic myeloid leukemias, or even a leukemoid reaction. Myeloblasts, monoblasts, and megakaryoblasts comprise the blast cell count. The FAB approach with additional criteria should be used to distinguish AUL and various subtypes of AML (Ml to M7 and M6Er) and to differentiate MDS, MDS-ER, chronic myeloid leukemias, and leukemoid reaction. Bone marrow core biopsy and electron microscopy may be required to confirm the specific diagnosis. Immunophenotyping with lineage specific antibodies is in its infancy in veterinary medicine. Development of this technique is encouraged to establish an undisputed identity of blast cells. Validity of the proposed criteria needs to be substantiated in large prospective and retrospective studies. Similarly, clinical relevance of cytomorphologic, cytochemical, and immunophenotypic characterizations of AML in dogs and cats remains to be determined.     

An improved ferritin assay for canine sera

An improved serum ferritin assay for canine serum has been developed. It uses two monoclonal antibodies in a sandwich arrangement. Serum ferritin can be determined on undiluted canine sera with this assay. The recovery of ferritin added to canine serum ranged from 98 to 106%, the within-assay coefficient of variability was 3.3 to 4.5%, and the assay-to-assay variability was 9.8 to 10.2%. Serum ferritin from 61 apparently healthy dogs had a geometric mean of 252 ng/ml, with a range of 80 ng/ml to 800 ng/ml.     

Calving patterns in seasonal dairy herds

Two field studies examined the calving patterns of cows in seasonal dairy herds in the Waikato (Field Study 1) and South Taranaki regions (Field Study 2). The first study examined patterns for cows commencing their second or subsequent lactation in herds which had used an inseminating service during the previous season. The second study included first lactation heifers only in 15 herds where animals had been naturally mated, and in 15 herds in which they had been synchronised and then artificially inseminated at the synchronised oestrus. The parameters describing calving patterns were based on the date for each herd's planned start of calving (PSC), which was 282 days from the date on which breeding commenced in the preceding season. The average interval from PSC to mean calving date for the 35 herds in Field Study 1 was 22 days, with individual herds ranging from 15 to 30 days. In herds with heifers which had been naturally mated (Field Study 2), it was 17.6 days compared to 11.0 days for previously synchronised animals. Calculating the intervals from PSC to median calving date and separately for the last two quartiles more effectively described a herd's calving pattern. The duration for the last quartile of the calving pattern was influenced by the extent and timing of induced calving. In Field Study 1, 88.6% of the 35 herd owners induced premature parturition in at least one cow. In these herds, 11.3% of cows were treated and calved prematurely. Only 61.7% of heifers which had previously been naturally mated calved by 3 weeks after PSC. Their calving dates were not evenly distributed over this 3-week period, with 9.8% in the first week and 25.6% in the third week. The calving pattern for heifers which had been previously synchronised showed several distinct peaks. Calvings to the synchronised mating were completed 15 days after PSC, by which time 64.7% of animals had calved. By 3 weeks after PSC, 72.9% of these heifers had calved. The results showed that there was considerable variation in calving patterns in seasonal dairy herds. This variation would have been due to differences in conception pattern, and the way induced calving had been applied. The calving pattern in heifers which had been naturally mated was less concentrated than had been expected. Synchronisation can significantly concentrate the calving pattern of these first lactation animals. The parameters used to describe calving patterns may be less applicable in herds in which a high proportion of animals is induced to calve prematurely, or where a whole herd is synchronised. Nonetheless, they do serve as an illustrative example of the variation in calving patterns among herds.     

Determination of uric acid in canine serum and urine by high performance liquid chromatography

A reproducible high performance liquid chromatography (HPLC) method was developed for analysis of uric acid in canine serum and urine. The method consists of precipitating serum proteins with phosphotungstic acid prior to HPLC analysis. Urine is analyzed after dilution with buffer. Chromatography is performed on a reversed-phase C-18 column with UV detection at 292 nm. Sensitivity of the method will allow reproducible measurement of uric acid at concentrations of 0.05 mg/dl in serum and 0.1 mg/dl in urine. The HPLC method has been used to quantify hundreds of canine serum and urine samples. The method is superior to UV absorption or colorimetric methods because its lower limit of detection allows measurement of uric acid at concentrations found in canine serum and urine.