The detection of capsaicin and dihydrocapsaicin in horse serum following long-term local administration.

BACKGROUND: Capsaicin and dihydrocapsaicin are alkaloids with analgesic effects in humans and animals. When used locally, both of them minimalise pain sensation by defunctionalising nerve endings. According to the Federation Equestrian International Prohibited Substances List, these are substance banned in horse competitions. The aim of the study was to determine the detection time of capsaicin in both plasma and serum after long-term use of a gel recommended for commercial use and applied as intended. The objective of the study was to select the best material for the detection of capsaicin as a doping substance in horses. METHODS: Nine healthy mature horses were administered 0.1% capsaicin topically in the form of a commercial analgesic gel (15 g of the gel per limb) to the front limbs every 24 hours for five days with a polar fleece bandage. Blood serum and plasma were collected prior to gel application and in the 12th, 18th, 24th, 36th, 42nd, 48th, 60th, 84th, 108th, 132nd, 156th hour after the gel application. Qualitative and quantitative analysis was performed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). RESULTS: The concentration of capsaicin in the serum samples did not exceed the lower limit of quantification. Capsaicin was not detected in the plasma samples during the entire study period. Dihydrocapsaicin was not detected in blood serum or plasma. CONCLUSION: The presented results suggest that capsaicin is not detected in horse serum in the 24-hour-periodfollowing its last application according to the dosage regimen used by owners and veterinarians for therapy rather than doping, based on a five day gel application and a polar bandage.

Conjunctival aerobic bacterial flora in healthy Silesian foals and adult horses in Poland.

BACKGROUND: Commensal bacterial and fungal flora of the conjunctival sac has been described in horses and other animals. The identification of commensal flora of the conjunctival sac may aid in the diagnosis of ocular inflammatory diseases, such as conjunctivitis or more severe ulcerative keratitis, common in horses. Moreover, damage of ocular protective barriers may lead to an opportunistic infection. The study was carried out in Silesian horses kept at a single breeding center in South-western Poland, in order to limit any breed-dependant and climate-dependant variables affecting the results. Following an ophthalmic examination that revealed no abnormalities, sterile swabs were collected from conjunctival sac in 26 adult horses and 11 foals. The obtained swabs were subjected to bacterial culture testing. In case of Staphylococcus spp. isolation, susceptibility to methicillin was evaluated. RESULTS: Forty- three bacterial isolates, representing eleven genera of bacteria were cultured from 30 (81%) horses. Gram-positive bacteria were the dominant isolates (72%) (p < 0.001). The most commonly isolated Gram-positive bacteria were Staphylococcus spp., while Moraxella spp. were the most frequently isolated Gram-negative bacteria. There was no significant influence of sex and age on the frequency and type of microbial isolates. CONCLUSIONS: Commensal flora is present in the conjunctival sac of healthy horses in Poland. Age does not affect the abundance and type of microbial isolates.

Morphology and elemental analysis of free range and stabled Polish Konik horses hair using Energy-dispersive X-ray spectroscopy (EDS).

Hair is a more biologically stable material than other tissues and contains a relatively constant level of minerals, drugs or toxins. The content of essential elements in hair depends on dietary and non-dietary factors. The aim of this study was to assess and compare the effect of diet on the elemental composition and morphological properties of the hair of healthy Polish Konik horses. Mane hair was collected from 19 horses living in The National Park in Popielno in Poland. Six of these horses were free-ranged all year with permanent access to pasture, while 13 horses were kept in the stable and fed oats, hay and straw. The samples were analyzed using a Scanning Electron Microscope combined with Energy-dispersive X-ray spectroscopy (SEM-EDS). Each hair was analyzed for its microscopic appearance and elemental Mg, Si, S, Ca, Mn, Co, Zn and Se content. No significant changes in the morphological properties and elemental composition were observed. There was inter- individual variability in element content between horses in both groups, but this did not affect their health. The comparison of the present results with other studies shows that the level of elements in the hair of Polish Konik horses is significantly lower than in healthy horses of other breeds. The traditional feeding plan containing pasture, oats and hay does not contain a sufficient amount of main mineral elements. Further investigation is needed to explain the effect of diet and maintenance on the mineral balance of horses.

Searching for ivermectin resistance in a Strongylidae population of horses stabled in Poland.

BACKGROUND: There are no available studies describing the possible resistance of strongyles to ivermectin in horses in Poland. One hundred seventy three horses from nine stud farms from South-Western Poland were studied. The effectiveness of ivermectin was studied on the 14th day after ivermectin administration using the fecal egg count reduction test, and a long-term observation of the egg reappearance period was carried out. The fecal study was carried out using a modified McMaster method, which typically detects 20 eggs per gram of stool. The results were subjected to statistical analysis that enabled quantification of the eggs in the stool samples. RESULTS: The study revealed high efficacy of ivermectin on the 14th day after administration without a shortening of the egg reappearance period. CONCLUSION: The results indicate that strongyles resistance to ivermectin in Poland is not a serious problem.

Prevalence and severity of ultrasonographic pulmonary findings in horses with asthma - a preliminary study.

Asthma is one of the most common non-infectious respiratory diseases in horses. Ultrasound examination is a widely available non-invasive additional diagnostic tool. To date, there are no studies focusing on ultrasonographic findings in horses with asthma. The aim of this study was to analyse the prevalence and severity of ultrasound lesions in lung tissue in horses with asthma. Lung ultrasonography was carried out on six healthy horses (controls) and 12 horses with asthma (six with mild and six with severe asthma). The sonographic changes in three lung sections were assessed using a scoring system. The most common changes present in all the animals were comet- tail artefacts. More advanced lesions were present in horses with severe asthma. Statistically significant differences in the overall average intensity of the ultrasound changes were seen between the controls and the study group and between the horses with mild and severe asthma. The lesions were usually located in the caudal lung regions, but they were also present in other areas as the disease progressed. Ultrasonography is a useful additional diagnostic tool enabling an assessment of the stage of the asthma progression. It is a very sensitive technique that visualizes minor lesions in the lung tissue even in clinically healthy animals. Due to its low specificity, it cannot replace endoscopy and the bronchoalveolar lavage in horses with asthma.

Assessment of sand accumulation in the gastrointestinal tract and its excretion with stool in silesian foals.

The aim of this study was to assess sand accumulation in the gastrointestinal tract and fecal sand excretion in Silesian foals using three diagnostic methods and taking into account the sex and age of the animals. Another aim of the study was to compare the three diagnostic methods. The study was carried out on 21 clinically healthy Silesian foals (10 females and 11 males) from 9-28 weeks old grazed on permanent pasture. The sand intake was assessed using a sedimentation test, abdominal ultrasonography and a quantitative evaluation of sand per 100 g of stool. In the sedimentation test, the sand was palpable in the stool of 57.1% of the horses, and clearly visible in 42.9% of the animals. The ultrasound examination revealed the presence of sand in the gastrointestinal tract in 66.7% of the horses. It was limited to a single location in 60% of the horses, while it was present in several regions in 40% of the horses. The mean amount of sand was 0.14 ± 0.33 g per 100 g of stool. It did not exceed 0.1g in 71.4% foals, while it ranged from 0.1-0.5 g in 23.8% foals. In 4.8% of the animals, it amounted to 1.6 g per 100 g of stool. There was no correlation between age and gender and the results. There was a positive correlation between the ultrasound examination and the sedimentation test. Sand may be accumulated in the gastrointestinal tract of foals without any clinical signs. The amount of sand excreted in the stool is not an indicator of the amount of sand accumulated in the gastrointestinal tract. An abdominal ultrasound examination should be combined with a sedimentation test for more specific results.

Right ventricular function during acute exacerbation of severe equine asthma.

BACKGROUND: Pulmonary hypertension has been described in horses with severe equine asthma, but its effect on the right ventricle has not been fully elucidated. OBJECTIVES: To evaluate right ventricular structure and function after a 1-week period of pulmonary hypertension secondary to acute exacerbation of severe equine asthma. STUDY DESIGN: Prospective study. METHODS: A clinical episode of severe equine asthma was induced experimentally in six susceptible horses. Examinations in remission and on day 7 of the clinical episode included a physical examination with clinical scoring, echocardiography, arterial blood gas measurements, venous blood sampling for cardiac biomarkers, intracardiac pressure measurements, right ventricular and right atrial myocardial biopsies, airway endoscopy and bronchoalveolar lavage. After 1 month of recovery, physical examination, echocardiography and cardiac biomarker analysis were repeated. Echocardiographic and pressure measurements were compared with those in 10 healthy control horses. RESULTS: All horses developed clinical signs of acute pulmonary obstruction. Right heart pressures increased significantly. Altered right ventricular function could be detected by tissue Doppler and speckle tracking echocardiography. Cardiac troponin concentrations did not increase significantly, but were highly elevated in one horse which exercised in the paddock prior to sampling. Focal neutrophil infiltration was present in two myocardial samples. Even in remission, asthmatic horses showed a thicker right ventricular wall, an increased left ventricular end-systolic eccentricity index at chordal level and decreased right ventricular longitudinal strain compared with controls. MAIN LIMITATIONS: The induced clinical episode was rather mild and the number of horses was limited because of the invasive nature of the study. CONCLUSIONS: Pulmonary obstruction in asthmatic horses induces pulmonary hypertension with right ventricular structural and functional changes.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

The micronucleus assay using peripheral blood reticulocytes from X-ray-exposed mice.

A new 'fluorescence' variant of the micronucleus assay using supravital staining of peripheral blood reticulocytes with an acridine orange coated slide was recently developed. In this study the application of this method to detect a mutagenic response to low-dose exposure of X-irradiation is reported. The mice were exposed to a single dose of 2.5, 5, 10, 15, 25, and 50 cGy. The induction of micronuclei in peripheral blood reticulocytes was recorded with sampling times of 0, 24, 48, 72, and 96 h after exposure without killing of mice. Dose-dependent effects were observed at sampling times of 24-72 h reaching maximum levels at 48 h after X-ray exposure. A highly significant (p < 0.01) increase of the micronucleated reticulocytes was found for doses of 10, 15, 25, and 50 cGy. Neither the dose of 2.5 nor 5 cGy induced the significant increase in the incidence of micronucleated reticulocytes compared with the respective control group.

[Evaluation of the usefulness of the peripheral test for determination of rabies vaccine potency]. / Ocena przydatnosci testu peryferyjnego do badania mocy szczepionki przeciw wsciekliznie.

The results of CDC peripheral test for potency of rabies vaccines were compared to the results obtained in the NIH intracerebral test recommended by the WHO. We did not find statistically significant differences in the potency of tested rabies vaccines by CDC and NIH tests.

[Effects of aluminum and deferoxamine on concentration of essential elements in animals experimentally intoxicated with aluminum]. / Wplyw glinu i deferoksaminy na zawartosc biopierwiastków u zwierzat doswiadczalnych poddanych intoksykacji glinem.

To assess the effect of aluminium intoxication in tissues of experimental animals on Ca, Mg, Zn, Cu, and Fe concentration, aluminium nitrate was administered intraperitoneally to mice at a daily dose of 0.27 mMol/kg for 5 weeks. Concentration of Al, Ca, Mg, Zn, Cu, and Fe were analyzed by atomic absorption spectrometry. The Al content in liver and tibia was significantly higher in treated mice in comparison with control group. In Al loaded tissues the significant increase of all tested essential elements was found. To evaluate the results of DFO treatment on essential elements, mice received 6 times intraperitoneally 3.5 mMol/kg of DFO. This treatment had generally no effect on reduction of Al concentration in tibia and liver, as well as on changes in essential elements concentration.

[Evaluation of contamination of serum derived intravenous preparations]. / Ocena stopnia zanieczyszczenia glinem wybranych preparatów osoczopochodnych do stosowania dozylnego.

The concentration of Al in 4 lots of 5% Albumina and in 14 lots of 5% immunoglobulins were estimated by adsorptive stripping voltammetric method. Mean Al concentrations amounted to 142.6 micrograms/l in 5% Albumina and to 202 micrograms/l in IVIG preparations produced in Poland. Al concentrations in foreign IVIG preparations ranged from 14 to 54 micrograms/l. These data show the need to consider setting an upper limit of microgram Al/l in i.v. drug products produced or registered in Poland.

[Heat stability of potency of bacterial vaccines and the titer of tetanus antitoxin of Polish production]. / Stabilnosc cieplna mocy szczepionek bakteryjnych oraz miano antytoksyny tezcowej produkcji krajowej.

Described the stability of potency vaccines (DTP, BCG) and immunoglobulins (human's and animal's) at storage and experimental temperatures. Thermal degradation rate and design of loss of potency in time have been determined by Arrhenius equation. Our results were similar to WHO data from preparations which have been made in another countries.

[Radiation induced adaptation in peripheral blood reticulocytes of mice]. / Indukcja popromiennej odpowiedzi adaptacyjnej w retikulocytach krwi obwodowej myszy.

An in vivo micronucleus assay in mice reticulocytes of peripheral blood for identifying the possibility of induction of adaptive response to various doses of radiation: 2.5 cGy or 5.0 cGy as adapting and 50 cGy or 100 cGy as challenging doses was performed. The most effective inhibition of frequency appearance of micronucleus in RETs of mice (i.e. adaptive response) takes place et the following experimental conditions: 5.0 cGy as adaptive dose and 50 cGy challenging dose. The interval between them were 4 hours. The maximum inhibition of frequency of micronucleus was et 24 h sampling time after exposure of challenging dose. This reduction was statistically significant (p < 0.05; p < 0.01-chi square test).

The level of endotoxin contamination in biopreparations.

Bacterial endotoxins as contamination of biopreparations have been estimated by chromogenic LAL test. Study on some compounds (aluminium hydroxide, formaldehyde and merthiolate) being components of vaccines showed no effect on the result of LAL test. The level of endotoxins in virus vaccines with the limits defined in producers certificate was adequate, the level of endotoxin was also low in virus vaccines of undefined requirements. The concentration of endotoxin in bacterial vaccines was differentiated. Considering the results of our experiments, as well as the fact, that the requirements for endotoxin contamination of bacterial vaccines are not available it seems necessary to establish the limits for these group of biopreparations.