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.     

Radio Frequency Signals in Jupiter's Atmosphere

During the Galileo probe's descent through Jupiter's atmosphere, under the ionosphere, the lightning and radio emission detector measured radio frequency signals at levels significantly above the probe's electromagnetic noise. The signal strengths at 3 and 15 kilohertz were relatively large at the beginning of the descent, decreased with depth to a pressure level of about 5 bars, and then increased slowly until the end of the mission. The 15-kilohertz signals show arrival direction anisotropies. Measurements of radio frequency wave forms show that the probe passed through an atmospheric region that did not support lightning within at least 100 kilometers and more likely a few thousand kilometers of the descent trajectory. The apparent opacity of the jovian atmosphere increases sharply at pressures greater than about 4 bars.     

Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) of Brucella abortus in cattle and mice

Brucella abortus vaccines composed of native cell envelopes or outer membrane proteins of smooth strain 2308 were compared with a vaccine (PG) composed of the insoluble residue of strain 2308 cell envelopes which had been extracted with hot sodium dodecyl sulfate. Vaccines were given by injection in an oil base adjuvant containing trehalose dimycolate and muramyl dipeptide or without adjuvant. Mice vaccinated with 30 micrograms native cell envelopes or PG and challenged 4 weeks later with virulent B. abortus strain 2308 displayed equivalent levels of protective immunity at 1 and 4 weeks post-infection. Heifers were vaccinated with 5 mg of antigens in adjuvant; PG was also administered without adjuvant. Humoral and cell mediated immune (CMI) responses were tested at monthly intervals. PG without adjuvant induced negligible immune responses. Native cell envelope antigens induced significantly higher titers of whole cell agglutinins over a 3-month period than did PG, although revaccination with PG in adjuvant enhanced the production of agglutinins and both vaccines induced antibodies to the O polysaccharide. Lymphocyte blastogenesis responses and delayed hypersensitivity reactions to porin and group 3 proteins were stimulated by both native and PG vaccines, and the magnitude of the responses did not differ significantly between the treatment groups. These vaccines were therefore comparable in their capacity to induce protective immunity in mice and CMI responses in cattle, whereas antibody responses induced by PG in cattle were generally lower. These findings provide a basis for evaluation of nonliving B. abortus vaccines in cattle.     

Enhanced Resistance to Sclerotium rolfsii in Populations of Alfalfa Selected for Quantitative Resistance to Sclerotinia trifoliorum

ABSTRACT Sclerotinia trifoliorum and Sclerotium rolfsii are pathogens for which similar mechanisms of parasitism have been proposed. This suggested that resistance to these pathogens may be related in a common host plant. This study was undertaken to determine whether selection for quantitative resistance to Sclerotinia trifoliorum in alfalfa also increases resistance to Sclerotium rolfsii as expressed in excised leaf tissues and whole plants. Resistance in excised leaf tissues was evaluated according to the rate of necrosis induced by Sclerotium rolfsii following inoculation with mycelium. Resistance to Sclerotium rolfsii in whole plants was evaluated according to their survival following crown inoculations. Three alfalfa populations previously selected from cv. Delta for quantitative resistance to Sclerotinia trifoliorum exhibited enhanced resistance to Sclerotium rolfsii, in comparison with Delta or with susceptible populations, in excised leaf tissues. When whole plants of Delta and two of these populations, Sclerotinia trifoliorum resistant (STR) and Mississippi Sclerotinia resistant (MSR), were inoculated with Sclerotium rolfsii at 3 to 8 weeks of age, significant (P = 0.01) differences in survival were attributed to plant age at inoculation and alfalfa populations. Survival of both MSR and STR was significantly (P = 0.05) greater than for Delta; the best differential results were obtained by inoculating plants 5 to 7 weeks old. To evaluate relationships of resistance to Sclerotinia trifoliorum and Sclerotium rolfsii over a broader genetic background, additional populations were selected for resistance to Sclerotinia trifoliorum from four other alfalfa cultivars by leaf-inoculation techniques, and this resistance was confirmed by whole-plant inoculations. In excised leaf tissues, all four of these populations also expressed enhanced resistance to Sclerotium rolfsii in comparison with either parent cultivars or populations of comparable size selected at random. These results establish that selection for quantitative resistance to Sclerotinia trifoliorum in alfalfa also confers enhanced resistance to Sclerotium rolfsii as expressed in excised leaf tissues and whole plants. Results suggest, therefore, that genes for quantitative resistance to Sclerotinia trifoliorum and Sclerotium rolfsii in alfalfa are likely to be synonymous, overlapping, or closely linked.     

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.     

Clinical and anatomical features of lymphosarcoma in 118 cats

Objective To determine patients' characteristics and anatomical distribution of lesions in cats with lymphosarcoma. Design Prospective multi-institutional study of naturally occurring feline lymphosarcoma. Methods Veterinarians in Sydney were provided with free diagnostic laboratory services for suspect cases of feline lym-phosarcoma. Lymphosarcoma was diagnosed based on physical findings, radiographic and/or ultrasonographic images and results of cytological or histopathological examination. When owners were not interested in pursuing an antemortem diagnosis, suspect cases were collected for necropsy. Patients' characteristics and physical findings were recorded. A modified scheme for anatomical classification of lesions was devised including a ‘mixed’ category for cases which involved two or more anatomical forms. Results One hundred and eighteen cases were accrued over an 18 month period. The median age was 120 months and range 5 to 212 months. Age distribution was bimodal, with a small peak for cats less than 24 months, and a normal distribution centred on 97 to 120 months. Eighty cats were domestic crossbreds, 22 were Siamese or Oriental cats (including crosses), 6 were Burmese, 5 were purebred longhairs and the remaining 5 were one of a number of purebred shorthaired breeds. In comparison to 1017 consecutive cases admitted to our hospital for conditions other than lymphosarcoma, Siamese/Oriental cats were over-represented amongst lymphosarcoma cases (P = 0.0006). Male cats were also over-represented, accounting for 72 of 118 cases (P = 0.05). Abdominal lymphosarcoma was the most common anatomical form (43 cats), followed by mixed (39), nodal (20), mediastinal (9) and atypical (involving non-lymphoid organs, 7) forms. When analysed for specific organ involvement, 29 (25%) had mediastinal involvement, 71 (60%) had abdominal involvement including 60 (51%) with involvement of the intestinal tract and/or mesenteric lymph nodes and 36 (31%) with bilateral renal involvement, and 47 (40%) had peripheral lymph node involvement. No case of primary lymphoid leukaemia was identified. A noticeable subgroup of cats younger than 24 months had involvement of the anterior mediastinum with or without concurrent enlargement of cervical or axillary lymph nodes; Siamese/Oriental cats were over-represented in this subgroup. Among cases with nodal involvement, lymph nodes of the head and neck were frequently involved, mandibular nodes most commonly, followed by superficial cervical nodes. In seven cases a solitary node was affected. Conclusions Compared with similar surveys overseas, our cats were older and male cats were over-represented. There was a notable subgroup of young cats with mediastinal involvement. Siamese/Oriental cats were over-represented in this subgroup as well as in the larger population of cats with lymphosarcoma. Compared with overseas surveys, renal involvement, mixed cases and atypical cases (including nasal lymphosarcoma) were more common. A new subcategory of nodal lymphosarcoma, with involvement restricted to node(s) of head and neck, was identified.     

In-vitro comparison of 3 knotting techniques for lateral fabellotibial suture stabilization

This study evaluated the biomechanical characteristics of a single self-locking knot (sSLK) and a double self-locking knot (dSLK) compared with the square knot (SQ) for stabilization of cranial cruciate ligament rupture. Each knot underwent monotonic tensile and cyclical loading. Starting tension, elongation, stiffness, and load to failure were all evaluated. A value of P < 0.05 was considered significant. Starting tension, overall stiffness, and load to failure were all significantly greater in both the sSLK and dSLK compared with the SQ. There was no difference in elongation among the knots. There were no significant differences in starting tension, elongation, stiffness, and load to failure between the sSLK and the dSLK. The self-locking knots were stronger and stiffer than the SQ; there is no biomechanical advantage in using the dSLK compared with the sSLK.     

Using a GnRH agonist to obtain an index of testosterone secretory capacity in the cockatiel (Nymphicus hollandicus) and sulphur-crested cockatoo (Cacatua galerita)

Objective   Validation of a stimulation test for determining the steroidogenic capacity of the parrot testis. The major aim was to characterise testosterone secretion after injection of a gonadotropin-releasing hormone agonist (GnRHa), then use the test to investigate seasonal reproduction in the male cockatiel.
Procedure   A synthetic GnRHa (buserelin; 8.0 µg of peptide/kg bodyweight) was injected IM into male cockatiels (n = 7) and sulphur-crested cockatoos (n = 3) and serial blood samples collected at 0, 30, 60, 90 and 120 min after administration. Once validated, the technique was subsequently used to examine seasonal changes (23 months) in the testosterone profile of a captive cockatiel population.
Results   Injection of buserelin resulted in a significant increase in the testosterone concentration of cockatiel plasma, with maximal concentrations occurring at approximately 60 (1.33 ± 0.08 ng/mL) to 90 min (1.22 ± 0.08 ng/mL) after injection. Although no clear pattern of seasonal variation in testosterone secretion was detected in cockatiel plasma, samples taken 60 and 90 min after administration showed a significant increase in all seasons. Injection of buserelin in the sulphur-crested cockatoo also resulted in increased testosterone secretion, with maximal concentrations obtained after 90 min.
Conclusion   Buserelin can be used to obtain a reliable index of the prevailing testosterone capacity of the cockatiel and cockatoo testis. With further studies, this test may be incorporated into clinical assessment of reproductive status.     

A framework for evaluation of on-farm mastitis diagnostics in Australia

Numerous culture-based diagnostics are available on the Australian and international markets for on-farm detection of bacterial pathogens in milk. Use of such diagnostics may provide an opportunity to improve the prudent use of antimicrobials in udder health management. Farms are low-resource settings in terms of diagnostic microbiology capacity. The World Health Organisation has identified criteria for the evaluation of diagnostic tests in low resource settings based on Accuracy, Sensitivity, Specificity, User-friendliness, being Rapid or Robust, Equipment-free and being Deliverable (ASSURED). Here, we review how those criteria can be interpreted in the context of microbiological diagnosis of mastitis pathogens, and how on-farm diagnostics that are currently available in Australia perform relative to ASSURED criteria. This evaluation identifies multiple trade-offs, both with regard to scientific criteria and with regards to convenience criteria. More importantly, the purpose of testing may differ between farms, and test performance should be evaluated relative to its intended use. The ability of on-farm mastitis diagnostics to inform mastitis treatment decision-making in a timely and cost-effective manner depends not just on test characteristics but also on farm-specific pathogen prevalence, and on the farm enterprise's priorities and the farm manager's potential courses of action. With most assay evaluations to date conducted in professional laboratories, there is a surprising dearth of information on how well any of the diagnostic tests perform on-farm and, indeed, of the on-farm decision-making processes that they aim to inform.