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.     

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).     

A Field Trial of a Pilated Moraxella bovis Bacterin for the Prevention of Infectious Bovine Keratoconjunctivitis

Two field trials were conducted to evaluate the efficacy of a Moraxella bovis bacterin in the control of infectious bovine keratoconjunctivitis. The bacterin did not affect the incidence of infectious bovine keratoconjunctivitis in either trial but did appear to reduce the severity of lesions, treatments required and withdrawals from pasture required in one trial. Possible reasons for the apparently contradictory results are offered. It is suggested that the bacterin may be of some value when used in conjunction with other preventive measures.     

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.     

Carbon storage by urban tree cultivars, in roots and above-ground

Urban trees can favorably affect factors underlying global warming by storing carbon and by reducing energy needs for cooling and heating buildings. To estimate carbon stored in roots and above-ground portions of trees, data was collected consisting of whole tree sampling of Amelanchier, Malus, Pyrus, and Syringa cultivars. Roots were excavated using an Air-Spade^™. Regression analysis resulted in two equations for predicting total carbon storage based on height and diameter of trees up to 20 cm dbh: Y = 0.05836 (dbh²) for root carbon storage, and Y = 0.0305 (dbh² × h)^0.9499 for above-ground carbon storage, explaining 97% and 96% of the variation, respectively. Average carbon stored in roots of various cultivars ranged from 0.3 to 1.0 kg for smaller trees, those 3.8 to 6.4 cm dbh, to more than 10.4 kg for trees 14.0 cm to 19.7 cm dbh. Average total carbon stored by cultivars ranged from 1.7 to 3.6 kg for trees less than 6.4 cm dbh to 54.5 kg for trees larger than 14.0 cm. The data from these equations apply mainly to trees in nurseries and recently transplanted trees. Comparisons showed that above-ground estimates from previous studies using a sampling technique overestimated values obtained from actual above-ground weights.     

Erysipelothrix septicemia in a little blue penguin (Eudyptula minor).

On June 25, 2002, aquarium veterinarians treated a 5-year-old, male little blue penguin (Eudyptula minor) that was acutely recumbent and dull, with inappetence of 24-hour duration. The penguin died within 10 minutes of presentation despite emergency resuscitation efforts. Gross pathologic findings consisted of pulmonary congestion and intestinal hemorrhage. Histopathologic findings included necrosis of tips of intestinal villi, increased numbers of mononuclear cells in pulmonary interstitium and hepatic sinusoids, and gram-positive bacteria in systemic microvasculature. Transmission electron microscopic examination revealed short gram-positive bacilli located in lumina of glomerular capillaries and in cytoplasm of mononuclear phagocytic cells in the lung and liver. Erysipelothrix rhusiopathiae was recovered from the lung, liver, and intestine by bacteriologic culture. Amplicons from polymerase chain reaction (PCR) tests using Erysipelothrix genus-specific primers and total genomic DNA extracted from formalin-fixed, paraffin-embedded tissue sections of lung and intestine demonstrated 99% nucleotide sequence identity with 16S small-subunit ribosomal DNA of E. rhusiopathiae and E. tonsillarum. The source of infection was speculated to be fish in the diet; however, repeated attempts to detect Erysipelothrix spp. from the mucous layer of food fish using bacteriologic culture and PCR were unsuccessful. This is the first report of erysipelas in a captive aquatic bird. Details of the isolation of E. rhusiopathiae and the application of molecular testing to identify Erysipelothrix DNA in formalin-fixed, paraffin-embedded tissue sections are given.     

Interfertility of Two Mating Populations in the Gibberella Fujikuroi Species Complex

Gibberella fujikuroi and Gibberella intermedia(mating populations ‘C’ and ‘D’ of the G. fujikuroi species complex) can be distinguished by differences in the spectrum of mycotoxins produced, the lack of sexual cross-fertility and diagnostic differences in their DNA sequences. Some isolates from these two biological species, however, can interbreed and complete meiosis to produce viable progeny. Analysis of marker segregation amongst such hybrid progeny can be used to estimate the degree of genomic rearrangement and genetic incompatibility that has accumulated since these sibling species diverged. Recombinant progeny were isolated from crosses of the standard tester strains for these two species and from crosses between these standard testers and a field isolate (KSU X-10626) that was cross-fertile with tester strains of both species. Progeny in all of the crosses segregated for amplified fragment length polymorphisms (AFLPs). Segregation of AFLP loci deviated from 1:1 for two thirds of the loci amongst the progeny of the cross between the ‘C’ and ‘D’ mating population tester strains, but <20% of the polymorphic loci in the cross of either tester with KSU X-10626 showed such distortion. It was concluded that G. intermedia and G. fujikuroi are sufficiently interfertile to belong to the same biological species, but that changing the nomenclature to reflect this interfertility requires more evidence for the natural occurrence of a continuum in fertility than is presently available.     

Clinical Evaluation of Tavocept to Decrease Diuresis Time and Volume in Dogs with Bladder Cancer Receiving Cisplatin

Background

Transitional cell carcinoma is the most common bladder cancer of dogs. Cisplatin combined with piroxicam provides superior response rates, but unacceptable rates of nephrotoxicity. Tavocept is a chemoprotectant that has mitigated cisplatin toxicity and decreased the required infusion/diuresis volume in clinical trials in humans.

Hypothesis/Objectives

We hypothesized that Tavocept would decrease diuresis volume and time and facilitate safe administration of a cisplatin/piroxicam protocol to dogs with bladder cancer. Secondary objectives were to compare response rate and survival times to an historical comparator group treated without Tavocept.

Animals

Fourteen client‐owned dogs were prospectively enrolled.

Methods

Tumor volume was measured by computed tomography at days 0, 42, and 84. Dogs received combination Tavocept/cisplatin with a shortened diuresis protocol. A total of 4 doses was planned, with concurrent administration of piroxicam. Serial biochemical analyses were evaluated for azotemia.

Results

A 90‐minute infusion/diuresis time was used for all dogs. Three dogs (21%) had concurrent increases in serum creatinine (>2.0 mg/dL) and BUN (>42 mg/dL) concentrations; 2 of these dogs were isosthenuric. This frequency of nephrotoxicity is significantly less (P = 0.0406) than that of an historical control group treated without Tavocept. Overall response rate was 27%. Median survival time was comparable to historical controls (253 vs. 246 days).

Conclusions and Clinical Importance

Tavocept decreased the required diuresis time with cisplatin from > 6 hours to 90 minutes, while also decreasing occurrence of azotemia. Survival time was comparable, but the response rate was inferior to an historical comparator group. Further evaluation in other tumors susceptible to platinum agents is warranted.