In vitro formation of metabolic-intermediate cytochrome P450 complexes in rabbit liver microsomes by tiamulin and various macrolides

Tiamulin and a number of macrolides were evaluated as to their ability in forming metabolic-intermediate (MI) complexes with cytochrome P450 in liver microsomes from rabbits bred for meat production. Complex formation, which occurred only in preparations where the expression of P450 3A was increased as the result of rifampicin pre-treatment and with different kinetics, was in the order tiamulin > erythromycin > TAO approximately roxithromycin approximately tylosin and did not take place with tilmicosin and spiramycin. Most of the tested compounds underwent an oxidative N-dealkylation and a good relationship could be found between the rate of N-dealkylase activity in induced preparations and the aptitude in generating MI complexes. Although the results from in vitro studies should be interpreted with caution, it is suggested that the potential for in vivo drug interactions also exists in the rabbit for tiamulin and for four out of the six tested macrolides.     

Adjuvants in veterinary vaccines: modes of action and adverse effects

Vaccine adjuvants are chemicals, microbial components, or mammalian proteins that enhance the immune response to vaccine antigens. Interest in reducing vaccine-related adverse effects and inducing specific types of immunity has led to the development of numerous new adjuvants. Adjuvants in development or in experimental and commercial vaccines include aluminum salts (alum), oil emulsions, saponins, immune-stimulating complexes (ISCOMs), liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, and a wide variety of bacterial derivatives. The mechanisms of action of these diverse compounds vary, as does their induction of cell-mediated and antibody responses. Factors influencing the selection of an adjuvant include animal species, specific pathogen, vaccine antigen, route of immunization, and type of immunity needed.     

Diversity of Epidemic Populations of Gibberella zeae from Small Quadrats in Kansas and North Dakota

ABSTRACT Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight (FHB) of wheat and barley and has been responsible for several billion dollars of losses in the United States since the early 1990s. We isolated G. zeae from the top, middle, and bottom positions of wheat spikes collected from 0.25-m(2) quadrats during severe FHB epidemics in a single Kansas (KS) field (1993) and in a single North Dakota (ND) field (1994). Three amplified fragment length polymorphism (AFLP) primer pairs were used to resolve 94 polymorphic loci from 253 isolates. Members of a subset of 26 isolates also were tested for vegetative compatibility groups (VCGs). Both methods indicated high levels of genotypic variability and identified the same sets of isolates as probable clones. The mean number of AFLP multilocus haplotypes per head was approximately 1.8 in each population, but this value probably underestimates the true mean due to the small number of samples taken from each head. Isolates with the same AFLP haplotype often were recovered from different positions in a single head, but only rarely were such apparently clonal isolates recovered from more than one head within a quadrat, a pattern that is consistent with a genetically diverse initial inoculum and limited secondary spread. The KS and ND samples had no common AFLP haplotypes. All G. zeae isolates had high AFLP fingerprint similarity (>70%, unweighted pair group method with arithmetic means similarity) to reference isolates of G. zeae lineage 7. The genetic identity between the KS and ND populations was >99% and the estimated effective migration rate was high (Nm approximately 70). Tests for linkage disequilibrium provide little evidence for nonrandom associations between loci. Our results suggest that these populations are parts of a single, panmictic population that experiences frequent recombination. Our results also suggest that a variety of population sampling designs may be satisfactory for assessing diversity in this fungus.     

Polymorphism of 14α-demethylase Gene (CYP51) in the Cereal Eyespot Fungi Tapesia acuformis and Tapesia yallundae

Cereal eyespot fungi Tapesia acuformis and Tapesia yallundae are closely related species which show different behaviours upon treatment with sterol 14-demethylase inhibitors (DMIs). T. acuformis is naturally resistant to DMIs belonging to the triazole family and susceptible to the imidazole ones, whilst T. yallundae is sensitive to both inhibitors. Cloning of the target enzyme gene, CYP51, from the two species revealed an important polymorphism between them. Further sequencing of CYP51 from sixteen T. acuformis and eleven T. yallundae strains with different phenotypes with regards to resistance to DMIs confirmed that at least eleven variations are species related. Among them, a conserved phenylalanine residue at position 180, found both in T. yallundae and in all known CYP51 proteins from filamentous fungi and yeast, was replaced in T. acuformis by a leucine. Therefore, a leucine at 180 could be possibly involved in natural resistance of T. acuformis to triazoles. Other mutations were observed in some resistant strains, sometimes simultaneously, but in contrast to what was reported for other filamentous fungi, where a mutation at the 136 position of the CYP51 gene product seemed to correlate with resistance to DMIs, we did not find a clear relationship between a given mutation and a particular phenotype. This result suggests that resistance to DMIs could have a polygenic nature in Tapesia. We took advantage of species-related variations to develop a PCR-based assay allowing rapid and easy discrimination between field strains of the two species.     

Detection of Salmonella organisms and assessment of a protocol for removal of contamination in horse stalls at a veterinary teaching hospital

OBJECTIVES: To assess methods of detecting environmental contamination with Salmonella organisms and evaluate a cleaning and disinfection protocol for horse stalls in a veterinary teaching hospital. DESIGN: Original study. SAMPLE POPULATION: 37 horses with diarrhea likely to be caused by Salmonella infection and their stall environments. PROCEDURES: Fecal samples were collected from horses daily during hospitalization; samples were obtained from stall sites after cleaning and application of disinfectants. Fecal and environmental samples were cultured for Salmonella spp and tested via polymerase chain reaction (PCR) assay to detect Salmonella DNA. RESULTS: 1 horse died and 2 were discharged prior to sample collection. Fecal samples from 9 of 34 horses yielded growth of Salmonella organisms on bacteriologic culture, and 23 yielded positive results via PCR assay on > or = 1 occasion. Among environmental samples from 21 stalls, salmonellae were detected at > or = 1 stall site on 6 of 78 occasions, and > or = 1 stall site yielded positive results via PCR assay on 69 of 77 occasions. Salmonella DNA was detected more frequently in samples of stall drains, cracks, and corners. Salmonella spp were cultured from samples of 3 stalls after both initial and second cleaning and disinfection cycles, but no organisms were detected in samples obtained after use of a peroxygen disinfectant. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that stalls in which horses with salmonellosis were housed should only be used to accommodate newly hospitalized horses after samples (collected after 2 cycles of cleaning and disinfection) from drains, cracks, and corners yield negative results on bacteriologic culture.     

Characterizing avian Escherichia coli isolates with multiplex polymerase chain reaction

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos,and the isolate of healthy birds that contained all the genes killed the most embryos amongthis group. These genes were not found among the non-E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.     

Effect of thyroid hormone supplementation on survival of euthyroid dogs with congestive heart failure due to systolic myocardial dysfunction: a double-blind, placebo-controlled trial

Nineteen euthyroid dogs of 12 breeds with echocardiographic signs of dilated cardiomyopathy (DCM) and radiographic and clinical signs of congestive heart failure (CHF) were evaluated in a randomised, double-blind, and placebo-controlled study. The dogs received either thyroxine or placebo as an adjunct to digoxin, furosemide and propranolol. The group assignment of individual dogs and serum concentrations of thyroid hormones remained unknown to owners and investigators during the entire study period. Dogs were evaluated clinically and with electrocardiography (ECG), thoracic radiography, echocardiography and measurement of total thyroxine (tT4) and thyroid stimulating hormone (TSH) before beginning of the trial, and then one week, 2 months, 6 months and yearly after initial examination, and, when applicable, at the time of euthanasia. End-point of the study was euthanasia (n = 17) due to severe congestive heart failure or sudden death (n = 2). Survival times ranged from 17 to 1030 days (median 187 days) in the placebo group, and from 18 to 1000 days (median 73 days) in the treatment group. There was no statistically significant difference in survival times between the treatment group and the placebo group (p = 0.46). Post mortem and histopathologic examinations revealed the attenuated wavy fiber type of DCM in 11 dogs, and myocardial infarcts, arteriosclerosis and chronic valvular disease in one dog. In conclusion, there was a wide range in survival times of dogs treated with digoxin, furosemide and propranolol. Adding thyroid hormones to the treatment did not significantly influence survival.