Evaluation of Cytotoxic Responses Caused by Selected Organophosphorus Esters in Chick Sympathetic Ganglia Cultures

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 × 10^-6M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 × 10^-3M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 × 10^2-M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 × 10^-2M (except triphenyl phosphate). Below 1 x 10^-7M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.     

Identification and characterization of bacteria isolated from crown galls on stone fruits in Poland

Eighty stone fruit nurseries located in different regions of Poland were examined for the presence of crown gall affected plants. The disease was observed in 39 nurseries, and galls were sampled for bacterial isolation. Out of 1213 isolates, 409 were pre‐identified as Agrobacterium/Rhizobium spp. with 23S rDNA‐based multiplex PCR, and out of these, 315 were pathogenic when tested on sunflowers. Sequence analysis of three housekeeping genes (fusA, recA, rpoD) revealed that 366 strains belonged to Rhizobium rhizogenes, 23 to Agrobacterium tumefaciens species complex, and the rest of the strains were allocated to new phylogenetic lineages. Of these, the most numerous was the lineage allocated in the Pararhizobium genus. Positive results obtained from pathogenicity tests were generally in agreement with results obtained by PCR with primers complementary to T‐DNA except for two strains, which were positive for PCR but negative for the pathogenicity test. All detected Ti plasmids were nopaline‐type. Independent of their pathogenicity, 59% of tested strains were not sensitive to agrocin 84 in in vitro tests. Analysis of biochemical and physiological features distinguished 50 groups with different phenotypic profiles, but the tested traits were not consistent for strains classified to one taxon. This finding shows limited value of biochemical tests in identification procedures. The bacteria causing tumours were heterogeneous and strains classified to different taxa were found even in a single tumour.     

Distribution of Salmonella serovars and phage types on 80 Ontario swine farms in 2004

The objective of this study was to describe the distribution of Salmonella spp. on Ontario grower-finisher pig farms. Eighty swine farms were visited from January through July 2004. On each farm, fecal samples were collected from 5 pens, 2 rectal samples and 1 pooled sample from fresh manure on the floor per pen. Salmonella was isolated from 91 (11%) of the 800 rectal samples and 73 (18%) of the 397 pooled samples. Overall, Salmonella was recovered from 37 (46%) of the 80 farms. On each positive farm, Salmonella was cultured from 1 to 7 pigs or 1 to 5 pens. Of the 37 farms, 18, 13, 5, and 1 yielded 1, 2, 3, and 4 serovars, respectively. The most common serovars were S. Typhimurium var. Copenhagen, S. Infantis, S. Typhimurium, S. Derby, S. Agona, S. Havana, and S. enterica subsp. I:Rough-O. The 3 most frequent phage types were PT 104, PT 104a, and PT 104b. There was a statistically fair agreement between samples collected directly from pigs and pooled pen samples in determining the Salmonella status at the pen and farm level (kappa = 0.6, P < 0.0001). However, in 62 pens, Salmonella status, serovars, or phage types differed between the pig and pooled pen samples. The distribution of Salmonella on the swine farms in this study indicates that, in developing an intervention strategy, priority should be given to farms positive for S. Typhimurium var. Copenhagen. Also, the variation in Salmonella status between pig and pooled pen samples deserves consideration in a sampling strategy.     

Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.     

Elimination of salicylic acid in goats and cattle

Sodium salicylate was administered to cattle and goats IV and PO according to a crossover design. Total urinary excretion of SA and its metabolites was measured for 3 days after dosing. Salicyluric acid (SUA) was the only metabolite detected in urine of either species. Recovery of sodium salicylate and SUA in goats amounted to 67.9 and 34.6% of the dose, respectively, after IV administration. After oral dosing, total recoveries were 30.2% (sodium salicylate) and 71.7% (SUA) of dose. By comparison, cattle excreted significantly (P less than 0.05) less sodium salicylate (54.0%) and more SUA (49.9%) after IV dosing. The same pattern was observed after oral administration, wherein cattle excreted less than 12% as sodium salicylate and more than 99% as SUA. In both species, almost 90% of the drug excreted as sodium salicylate was found in urine within the first 12 hours after an IV dose and within 24 hours after oral dosing. The excretion of SUA was somewhat slower in both species, especially after oral administration. The data suggested that there were only quantitative differences in the metabolism and elimination of sodium salicylate between the 2 species, with cattle excreting a higher proportion of the drug as the glycine conjugate SUA.     

Biofilm bacteria: formation and comparative susceptibility to antibiotics

The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined. Bacterial biofilms were readily formed on the CBD under selected conditions. The biofilms consisted of microcolonies encased in extracellular polysaccharide material. Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations. Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most effective antibiotics against E. coli growing as a biofilm. Salmonella spp. and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin. Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested. The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.     

Study of intracellular ion composition of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> biomass

The ionic composition of the of the intracellular content of the alcohol yeast Saccharomyces cerevisiae R. 1039 biomass was studied for confirmation of their use perspectiveness as a mediator to enable the management the mineral content of food in the manufacture of food and feed additives. The ionic composition of the obtained extracts were identified using the method of capillary electrophoresis. It is found that the quantitative ion content in the cell extracts depends on the concentration of the nutrient medium. When the yeast S. cerevisiae R. 1039 was cultivated on the medium with the soluble solids concentration of 30%, the intracellular ion content in the extracts was 1.3 times higher than when the yeast was cultivated on 12% wort by increasing the concentration of chlorides, sulfates, formates, potassium ions, and calcium. The yield of the yeast S. cerevisiae R. 1039 biomass increased 1.6 times per unit of volume of the medium with increase of the soluble solids concentrations from 12% to 30%.     

Comparison of anthelmintic activity of pyrantel, praziquantel, and nitazoxanide against Anoplocephala perfoliata in Horses

Three anthelmintics were compared for efficacy in reducing the egg production of Anoplocephala perfoliata in a herd of central Texas horses. Two trials were run, 1 in mares and the other in weanlings that were diagnosed as being infected with Anoplocephala by recovery of eggs in 5 g of feces with sugar centrifugation. Each animal was evaluated twice before treatment and again twice following treatment (at weeks 2 and 4 after treatment). The criteria for infection were the recovery of eggs on at least 1 occasion before treatment and the finding of eggs on 1 day following treatment. The mares were treated 1 time with either pyrantel pamoate at 13.2 mg/kg, nitazoxanide at 100 mg/kg, praziquantel at 1.23 mg/kg or remained as untreated controls. The weanlings were treated with pyrantel at 13.7 mg/kg nitazoxanide at 100 mg/kg or remained as untreated controls. The percentage reduction of patient infection in mares after treatment with pyrantel was 83%, with nitazoxanide was 78%, and with praziquantel was 83% and in controls was 17%. There was a 75% reduction of patient weanlings treated with pyrantel or nitazoxanide and a 17% reduction in untreated controls. The reduction of infection in all horses treated with any drug was significantly different from controls. All of the drugs were somewhat effective in the control of Anoplocephala, and there were no differences among the drugs in their effectiveness.

Introduction

Anoplocephala perfoliata, the lappeted tapeworm, is an inhabitant of the intestine of equids. Adult tapeworms attach to the intestinal mucosa at the ileocaecal valve and, when present in large numbers, cause edema and hypertrophy of the ileum. The disease manifest by this infection may be inapparent or may give rise to colic (abdominal pain) in the horse apparently from mechanical obstruction or intussusception of the small intestine into the caecocolon.^{1, 2, 3, 4, 5, 6, 7 and 8} The prevalence of infection is geographically variable^{9, 10, 11, 12 and 13} but appears to be increasing,¹⁴ with a much higher rate of infection found with necropsy as opposed to fecal observations. Horses become infected by the ingestion of infected orbatid mites in pastures. Orbatid mites, the intermediate hosts, are predatory and are found in decaying organic material, such as leaf litter. Horses of all ages are infected, but there are lower numbers of clinical cases in horses older than 4 years of age.⁴ The intensity of infection is highest in the late summer and autumn.^{8 and 12} Anthelmintics with reported efficacy against A perfoliata include pyrantel pamoate at 13.2 mg/kg,¹⁰ pyrantel tartrate at 2.6 mg/kg for 30 days,¹⁵ pyrantel embonate at 38 mg/kg,¹⁶ and praziquantel at 1 to 2 mg/kg.^{17 and 18} Nitazoxanide has not been evaluated for Anoplocephala but was included in the trial because of its effects against nematodes and tapeworms in humans.¹⁹ Because Anoplocephala infections may cause disease and there is a perception that current anthelmintics may not be as effective as in the past, a study was done to compare anthelmintics to lower the intensity of fecal egg counts in a herd of horses in central Texas.

Materials and methods

Quarter horse mares and weanlings from a single herd were evaluated with 5 g of feces with a sucrose double centrifugation test to determine whether eggs of Anoplocephala were present.²⁰ Feces from each individual horse were evaluated twice, once approximately 2 weeks before treatment and again on the day of treatment. If Anoplocephala eggs were found on either date, the horse was considered to have positive results. Within each group (mares or weanlings), the treatment selection was randomly allocated as the horses were restrained for treatment. Fecal samples were again evaluated at 14 and 28 days after treatment for the presence or absence of eggs on either day.The dose for each individual horse was determined by chest girth weight tape at the time of treatment. The treatments were as follows: pyrantel pamoate (Strongid-T, Pfizer Animal Health, Exton, Pa) at 13.7 mg/kg via nasogastric intubation (12 mares, 8 weanlings), nitazoxanide oral paste (Nitazoxanide, Idexx Laboratories, Westbrook, Me) at 100 mg/kg (9 mares, 8 weanlings), praziquantel (Droncet injectable, Bayer Corp, Shawnee Mission, Kan) at 1.23 mg/kg via nasogastric intubation (6 mares), and untreated controls (6 mares, 6 weanlings). A 1-tailed Fisher exact test was used to compare rates of infection before and after treatment. If a mare or foal did not have positive results before treatment, it was not evaluated in this study.

Results and discussion

No abnormal clinical signs were seen after treatment with any of the products. Treatment was administered to several additional animals with each product, but they were not included in the analysis if they did not have positive results on 1 of the 2 evaluations before treatment, hence, the different numbers of horses in treatment groups.None of the horses in the trial exhibited clinical signs associated with the infection of A perfoliata. However, before the trial, a mare from the infected herd exhibited signs of colic and Anoplocephala eggs were detected in the feces. Examination of the remainder of the herd gave impetus to the study.Mean egg counts before and after treatment are given in the Table.The presence of strongylate and Parascaris eggs in weanlings served as a control of the methodology of evaluation. The difficulty of finding Anoplocephala eggs has been recognized by several authors,^{5, 8, 13, 14 and 21} but the authors also recognize that when there were greater numbers of parasites there was increased egg production. Therefore, finding of eggs with fecal flotation indicated that there were 20 worms or more. However, there appears to be no correlation between the number of worms and egg counts once the detection threshold is reached,²² so the criterion for evaluation was the presence of eggs in the feces before treatment compared with after treatment. Although mean egg counts were not compared, the number of eggs in each infected horse was less after treatment in all groups compared with untreated controls (Table). The method of evaluation used in this study cannot be equated to those of critical^{10 and 16} or control¹⁴ studies in which horses are killed so that all worms are detected. However, the use of clinical studies to compare compounds is useful in detecting which anthelmintics are likely to be of value against geographically distinct populations of worms. Admittedly, more sampling may have increased the number of horses with positive results, both before and after treatment.