Bridging of a Liquid Chromatography-Tandem Mass Spectrometry Method for Oxytetracycline, Chlortetracycline, and Tetracycline in Bovine Kidney with the Official Microbial Growth Inhibition Assay.

BACKGROUND: Oxytetracycline (OTC), chlortetracycline (CTC), and tetracycline (TC) are approved antibiotics used to treat bacterial infections in cattle. To ensure human food safety, a tolerance has been established for the sum of these three TC residues as 12 parts per million in bovine kidney in the United States The current official regulatory method for quantifying these antibiotics in the target organ is a labor-intensive microbiological assay. OBJECTIVE: Our laboratory developed and validated a fast, selective, and less laborious method utilizing LC-tandem mass spectrometry for the determination and confirmation of the three tetracyclines (TET) in bovine kidney. METHODS: Briefly, homogenized kidney tissue was spiked with an internal standard (ISTD), and then was extracted with 1% phosphate buffer. The crude extract was cleaned up using solid-phase extraction cartridges before instrumental analysis. RESULTS: Accuracies for quantifying these three drugs in fortified kidney homogenate were between 99.9 and 110% at multiple concentrations, with respective CVs all below 9.5%. Quantitative correlation between the two methods (bridging) was evaluated with incurred bovine kidney samples for each of the three tetracyclines separately. The results were statistically evaluated using a measurement model called Functional Relationship Estimation by Maximum Likelihood. CONCLUSION: A linear quantitative relationship was demonstrated between the two methods within the concentration range of regulatory relevance. HIGHLIGHTS: This instrumental method is in addition to the established microbial assay for the detection of tetracyclines residue in beef kidney to ensure the food safety of cattle products.

Minimally invasive ultrasound-guided technique for central venous catheterization via the external jugular vein in pigs.

OBJECTIVE: To describe an ultrasound-guided technique for central venous catheter placement via the external jugular vein (EJV) in pigs. ANIMALS: 96 healthy Landrace-Poland China barrows (approx 16 weeks old with a mean weight of 70 kg). PROCEDURES: Pigs were anesthetized. With ultrasound guidance, a needle was inserted into the EJV without a large incision or cutdown procedure. A guidewire was inserted through the needle into the vein. A modified Seldinger technique was used to advance a catheter into the vessel until the tip was in the cranial vena cava near the right atrium. A trocar was used to create a tunnel through the subcutaneous tissues from the catheter insertion site to between the dorsal borders of the scapulae. The free end of the catheter was passed through that tunnel. An extension was attached to the catheter and secured to the skin. Pigs were euthanized and underwent necropsy at completion of the study for which they were catheterized. RESULTS: Central venous catheters were successfully placed in all 96 pigs and facilitated collection of serial blood samples with minimal stress. Catheters remained in place for a mean of 6 days (range, 4 to 10 days). Necropsy revealed abscesses along the subcutaneous catheter tract in 9 pigs. Twenty pigs had histologic evidence of phlebitis and fibroplasia in the cranial vena cava. CONCLUSIONS AND CLINICAL RELEVANCE: The described technique, in combination with extensive socialization, allowed serial collection of blood samples with minimal stress and restraint and is an alternative to surgical cutdown procedures for catheter placement.

Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Qualitative Sybr Green real-time detection of single nucleotide polymorphisms responsible for target-site resistance in insect pests: the example of Myzus persicae and Musca domestica.

Chemical insecticides have been widely used to control insect pests, leading to the selection of resistant populations. To date, several single nucleotide polymorphisms (SNPs) have already been associated with insecticide resistance, causing reduced sensitivity to many classes of products. Monitoring and detection of target-site resistance is currently one of the most important factors for insect pest management strategies. Several methods are available for this purpose: automated and high-throughput techniques (i.e. TaqMan or pyrosequencing) are very costly; cheaper alternatives (i.e. RFLP or PASA-PCRs) are time-consuming and limited by the necessity of a final visualization step. This work presents a new approach (QSGG, Qualitative Sybr Green Genotyping) which combines the specificity of PASA-PCR with the rapidity of real-time PCR analysis. The specific real-time detection of Cq values of wild-type or mutant alleles (amplified used allele-specific primers) allows the calculation of ΔCqW-M values and the consequent identification of the genotypes of unknown samples, on the basis of ranges previously defined with reference clones. The methodology is applied here to characterize mutations described in Myzus persicae and Musca domestica and we demonstrate it represents a valid, rapid and cost-effective technique that can be adopted for monitoring target-site resistance in field populations of these and other insect species.

Plasma pharmacokinetics of ceftiofur metabolite desfuroylceftiofur cysteine disulfide in holstein steers: application of nonlinear mixed-effects modeling.

Eight clinically normal and drug-naïve Holstein steers were dosed with ceftiofur sodium at 2.2 mg/kg body weight intramuscularly. Doses were given at 24-h intervals for 5 days. Prior to the first dose and after all injections, blood samples were collected serially for determination of plasma concentrations of one of ceftiofur's main metabolites, desfuroylceftiofur cysteine disulfide (DCCD). A nonlinear mixed-effect model was used to analyze the plasma concentration data. A stochastic approximation expectation maximization (SAEM) algorithm in MONOLIX version 4.2.2 was used to approximate the likelihood of the nonlinear mixed-effect model and to estimate the population parameters. In addition, simulation studies were conducted to justify the model and demonstrate how to interpret the model parameters given different scenarios.

Detection and survival of Toxoplasma gondii in milk and cheese from experimentally infected goats.

The consumption of unpasteurized goat cheese and goat's milk has been suggested as a risk factor for toxoplasmosis in humans. In the present study, detection and survival of Toxoplasma gondii in milk and cheese was studied by bioassay in mice (milk) and in cats (cheese). Eight goats were inoculated orally with 300 to 10,000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days postinoculation and bioassayed in mice and cats. For mouse bioassay, 50 ml of milk samples were centrifuged, and the sediment was inoculated subcutaneously into mice. Mice were tested for T. gondii infection by seroconversion and by the demonstration of parasites. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. For cat bioassay, 400 ml (100 ml or more from each goat) of milk from four goats from 6 to 27 days postinoculation were pooled daily, and cheese was made using rennin. Ten grams of cheese was fed daily to four cats, and cat feces were examined for oocyst shedding. One cat fed cheese shed oocysts 7 to 11 days after consuming cheese. Attempts were made to detect T. gondii DNA in milk of four goats; T. gondii was detected by PCR more consistently, but there was no correlation between detection of viable T. gondii by bioassay in mice and T. gondii DNA by PCR. Results indicate that T. gondii can be excreted in goat's milk and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk also should not be consumed.

A Holstein cow-calf model for the transfer of ciprofloxacin through milk after a long-term intravenous infusion.

This study is part of an ongoing effort to develop animal models that provide milk and sufficient infant (offspring) plasma samples to fully describe a drug's pharmacokinetics to quantitate the risk to the nursing infant. Ciprofloxacin was administered to six healthy Holstein cows as a constant rate intravenous infusion (flow rate was weight adjusted) to achieve a steady-state concentration of approximately 300 ng/mL for 7 days. Plasma and milk samples were collected from the cow at regular intervals over the course of the 7 days. The plasma and milk samples were analyzed for ciprofloxacin by high-performance liquid chromatography. The milk was fed to calves, and calf plasma samples were analyzed to study the lactational transfer of ciprofloxacin from dam to nursing neonate. Remarkably, concentrations of ciprofloxacin in milk were 45 times higher than plasma drug concentrations in the dam. Approximately 6% of the administered dose was transferred to the milk, resulting in an average oral dose of 0.5 mg/kg to the calves with every feeding. The drug did not accumulate in the calves, and plasma concentrations were between one-tenth and one-fifth the plasma concentrations of the dam.

Tissue/fluid correlation study for the depletion of sulfadimethoxine in bovine kidney, liver, plasma, urine, and oral fluid.

Sulfonamides are among the oldest, but still effective, antimicrobial veterinary medicines. In steers and dairy cows, the sulfonamides are effective in the treatment of respiratory disease and general infections. Sulfadimethoxine (SDM) has been approved by US Food and Drug Administration (FDA) for use in steers and dairy cows with a tolerance of 100 ng/g (ppb) in edible tissues and 10 ppb in milk. The detection of SDM residue above tolerance in the animal slaughtered for food process will result in the whole carcass being discarded. This report describes a comprehensive depletion study of SDM (and its main metabolite) in plasma, urine, oral fluid, kidney, and liver. In this study, nine steers were injected intravenously with the approved dose of SDM; the loading dose was 55 mg/kg, followed by 27.5 mg/kg dose at 24 h and again at 48 h. Fluids (blood, urine, and saliva) and tissue (liver and kidney) samples were collected at intervals after the last dose of SMD. The combination of laparoscopic serial sampling technique with the liquid chromatography/mass spectrometry method provided the data to establish the tissue/fluid correlation in the depletion of SMD. A strong correlation and linearity of the log-scale concentration over time in the depletion stage has been confirmed for kidney, liver, and plasma.

La determinación de PTH intraquirúrgica o posquirúrgica por quimioluminiscencia predice la hipocalcemia en el posoperatorio de la tiroidectomía en niños / Determination of PTH during or post surgery for quimioluminiscency can predict hypocalcemy in the post surgery children´s thyroidectomy

Objetivo: Evaluar la eficacia diagnóstica de la determinación de PTH en muestras de plasma tomadas durante la cirugía (intra) y posquirúrgica inmediata para predecir el riesgo de desarrollar hipocalcemia en el postoperatorio de la tiroidectomía total en pacientes pediátricos. Métodos: Se llevó a cabo un estudio de cohortes,prospectivo, longitudinal con 20 pacientes pediátricos en los que se practicó tiroidectomía total. Se determinaron los niveles de PTH preoperatorios, intraoperatorios y en el período posquirúrgico inmediato (basal, 5 y 60 minutos de la remoción de la glándula tiroides) utilizando un ensayo automatizado quimioluminiscente (IMMULITE, Siemens), límite de cuantificación 8 pg/mL, CV intra e interensayos < 5,4%. Para este estudio, la concentración de PTH de cada paciente no fue conocida por el equipo tratante hasta el final del mismo. Además se determinó la concentración en suero de Calcio total (Ca T) y/o Calcio iónico (Cai) regularmente durante las 48 hs posquirúrgicas y se controló la presencia de síntomas o signos de hipocalcemia. Se consideró hipocalcemia Ca T < 8 mg/dl y/o Cai < 0,8 nmol/L. Se realizó un análisis por curva ROC para determinar el nivel de PTH que fuera más eficaz en predecir la aparición de hipocalcemia según su sensibilidad (S), especificidad (E), eficiencia diagnóstica (ED) y Valor Predictivo Positivo (VPP). Resultados: Diez de los 20 pacientes (50%) desarrollaron hipocalcemia y 3 de ellos presentaron síntomas. La presentación de hipocalcemia sucedió: 40% en las primeras 6 hs y 40% a las 24 hs.vel de PTH en la muestra intraoperatoria < 14 pg/ml mostró S: 80%, E: 100%, ED: 90% (IC95%: 73-100) y VPP: 100% para predecir hipocalcemia posquirúrgica. En la muestra posquirúrgica inmediata, la concentración de PTH < 14 pg/ml presentó S: 80%, E: 90%, ED 82% (IC95% 63-100) y VPP 90% para predecir hipocalcemia posquirúrgica. Cuando la PTH intraquirúrgica o posquirúrgica es <14 pg/ml el riesgo relativo de presentar hipocalcemia postiroidectomía es de 9. Conclusiones: La medición de PTH intraquirúrgica y posquirúrgica es una herramienta eficiente para predecir hipocalcemia posquirúrgica por tiroidectomía total en la población pediátrica. Esta detección permite la inmediata decisión sobre el tratamiento suplementario con calcio en los pacientes de riesgo mejorando su evolución y evitando la presentación de tetania y otros síntomas de hipocalcemia. Además, permitiría disminuir los controles en los pacientes que evolucionarán con normocalcemia, reduciendo en ambos grupos de pacientes los costos de internación.

Efficiency of drug delivery to the coronary arteries in swine is dependent on the route of administration: assessment of luminal, intimal, and adventitial coronary artery and venous delivery methods.

PURPOSE: To compare the efficiency of five different drug delivery methods to the coronary artery in swine. MATERIALS AND METHODS: A nanoparticle-albumin-bound, nonradioactive isotopic marker was administered within the left anterior descending coronary artery (LAD) through a microinfusion catheter (MIC: adventitial, n = 8, and luminal, n = 4), a porous drug infusion balloon (DIB: intimal, n = 4), and a straight catheter (SC: luminal, n = 2) and within the superior vena cava (SC: intravenous, luminal, n = 2). The distribution of the marker in heart, lung, liver, kidney, muscle, blood, urine, and bile was determined 68-84 minutes after delivery. The heart was sectioned into six axial slices and each slice divided into four quadrants. The marker content was assayed by neutron bombardment and the total counts of disintegrations per minute (DPM) expressed as a percentage of the control for each device delivery control. RESULTS: After luminal delivery with the nonactuated MIC (MIC-NA) or intimal delivery with the DIB, 0.17% ± 0.07 and 0.39% ± 0.09, respectively, less than 0.39% of the total marker was detected in the heart. After adventitial delivery with the actuated MIC (MIC-A), 63.1% ± 9.9 of the total marker was detected in the heart. Marker was only detected in quadrants containing the coronary LAD, with the highest level in the middle slice and lower marker levels in consecutive proximal and distal heart slices. The nonactuated MIC-NA and DIB drug infusion balloon patterns of marker distribution were similar to those of actuated MIC-A, although with reduced levels. These delivery methods were also associated with considerably more marker detected in the lungs and liver: at least 22% compared with 1.34% ± 1.34 for the actuated MIC-A There was one delivery failure with the actuated MIC. CONCLUSIONS: Catheter-based adventitial delivery with the MIC-A represents a more efficient delivery method for retention of vascular therapeutics.

Isobaric (gasless) laparoscopic liver and kidney biopsy in standing steers.

The purpose of the current study was to investigate the suitability of an isobaric laparoscopic procedure, using a single port, for obtaining serial kidney and liver biopsy samples from standing steers. The samples were used in support of a pharmacokinetic tissue-fluid correlation study. Laparoscopic access was performed 3 times in each of 8 healthy Holstein steers, alternating from the right side to the left side and then to the right side again. The surgery was performed in standing stocks after the animals were given 3 doses of sulfadimethoxine sulfate intravenously and fasted for at least 18 h. Sedation and analgesia were achieved with acepromazine and xylazine. Lidocaine 2% was injected at the center of the paralumbar fossa (left or right), and an incision was made for introduction of a trocar-cannula assembly. Room air was allowed to enter the abdomen through the cannula at the time of insertion. Once the peritoneal cavity was reached, an operating endoscope was inserted. No pressurized insufflation was performed. A biopsy forceps was introduced into the operating channel of the endoscope to obtain a 100-mg kidney or liver sample. No complications were encountered. The 24 laparoscopic procedures provided 24 kidney and 16 liver samples. The results suggest that the isobaric (gasless) single-port laparoscopic technique is feasible for kidney and liver biopsy on standing steers. The procedure can be performed in a reliable and efficient manner in the sedated standing bovine.

Determination of sulfadimethoxine and 4N-acetylsulfadimethoxine in bovine plasma, urine, oral fluid, and kidney and liver biopsy samples obtained surgically from standing animals by LC/MS/MS.

A quantitative method was developed and validated to measure the concentration of sulfadimethoxine (SDM) and its major metabolite, (4)N-acetylsulfadimethoxine (AcSDM), in bovine tissues and body fluids. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) gave quantitative results for these two analytes in extracts from bovine plasma, urine, oral fluid, kidney, and liver, using SDM-d(4) as internal standard (I.S.). The lower limit of quantitation (LLOQ) for both analytes in these matrices was validated at 2, 100, and 5 ng/mL in plasma, urine, and oral fluid respectively, and 10 ng/g in both kidney (cortex) and liver. The overall accuracy (average of 4 levels) is, for plasma, 104% (SDM) and 95% (AcSDM), with standard deviation of 9% (SDM) and 15% (AcSDM); for urine, 100% (SDM) and 106% (AcSDM), with standard deviation of 5% (SDM) and 6% (AcSDM); for oral fluid, 103% (SDM) and 103% (AcSDM), with standard deviation of 4% (SDM) and 4% (AcSDM); for kidney, 101% (SDM) and 111% (AcSDM), with standard deviation of 7% (SDM) and 6% (AcSDM); and for liver, 99% (SDM) and 115% (AcSDM), with standard deviation of 11% (SDM) and 9% (AcSDM). C18 SPE cartridges were used to clean-up these matrices, except for urine which was diluted directly with buffer before analysis by LC/MS/MS.

Bovine kidney tissue/biological fluid correlation for penicillin.

Penicillin is one of the most commonly misused drugs in steers and dairy cows. In the US, at slaughter the tolerance is 50 ng/g in kidney and other edible tissues. If the tolerance is exceeded, the carcass may not be used for human food. A preslaughter test for penicillin in an easily accessible biological fluid is needed to predict if the concentration of penicillin is below tolerance in the kidney before the bovine is slaughtered. In this study, 12 steers were injected three times with the approved dose (7000 IU) of penicillin at 12-h intervals. Blood and urine samples were collected at intervals after the final dose of penicillin. At each sampling point, one kidney biopsy sample was collected by laparoscopic surgery in the live animal. Another kidney sample was collected at slaughter. Correlations between plasma and kidney concentrations and between urine and kidney concentrations were determined. These correlations predict with 95% confidence that 99% of the animals will have kidney tissue below penicillin tolerance when the plasma concentration of penicillin is below 0.4 ng/mL and/or the urine penicillin concentration is below 140 ng/mL.

Development of a technique for serial bilateral renal biopsy in steers.

A biopsy procedure was developed to provide serial kidney samples from standing steers. Ten clinically normal steers were given intramuscular injections of gentamicin sulfate, 4 mg/kg body weight. Renal biopsy was performed at 5 separate times. After feed was withheld for 24 h, laparoscopic surgery was performed in standing stocks. Acepromazine, xylazine, and butorphanol were used for sedation and analgesia, and 2% lidocaine was used for local anesthesia. Two incisions approximately 2 cm long were made in the paralumbar fossa to allow for trocar introduction. The abdomen was insufflated with CO2 and, with endoscopic guidance, a biopsy forceps used to remove a kidney sample 2 to 3 mm in diameter, by either a left or a right abdominal approach. Each operation was recorded on videotape, and images were also captured with a digital medical device system. Respiration, heart rate, temperature, appetite, attitude, and postural positions were evaluated at 12, 24, 48, and 72 h after surgery. The 51 laparoscopic procedures provided 48 renal samples (approximately 100 mg each). The 1st and 2nd samples were from the right kidney, and the 3rd sample was from either the left or the right kidney; the 4th and 5th samples were from the left kidney. Adhesions made an approach from the right side difficult for the 3rd sample. No clinical changes were observed in 9 steers after the procedure. One steer died after the 3rd procedure owing to hemorrhage.

Use of tissue-fluid correlations to estimate gentamicin residues in kidney tissue of Holstein steers.

Gentamicin continues to be one of the most effective antibiotics for the treatment of gram-negative infections. Greater than 90% of the drug is rapidly eliminated from the body in <2 days, however, a small residue remains bound to the kidney cortex tissue for many months. In beef steers, the gentamicin residue is unacceptable and its presence is monitored by the FAST (Fast Antimicrobial Screen Test) applied to the kidney at the time of slaughter. The sensitivity of the FAST to gentamicin in the kidney cortex is reported to be 100 ng/g, therefore, this level of gentamicin defines the acceptable limit of gentamicin drug residue in the bovine kidney. In the present study, three doses of 4 mg/kg gentamicin was administered intramuscularly to eight steers. Gentamicin was allowed to deplete from the kidneys for a range of times from 7 to 10 months. At slaughter the level of gentamicin in the kidney cortex varied from 91 to 193 ng/g, but a total of 160 FAST tests performed on the kidneys were negative. Blood and urine samples were collected at varying times following the last dose of gentamicin. Kidney tissue samples were collected by laparoscopic surgery in the live steers as well as the final sample obtained at slaughter. Plasma levels of gentamicin declined rapidly to nondetectable within 3 days, while measurable urine persisted for 75 days before the concentration of gentamicin declined to levels too low to quantitate by the available liquid chromatography tandem mass spectrometry (LC/MS/MS) technique. An estimated correlation between an extrapolation of urine gentamicin concentration to the corresponding kidney tissue sample suggests a urine to kidney tissue relationship of 1:100. A test system sufficiently sensitive to a urine gentamicin concentration of 1 ng/mL will correlate with the estimated 100 ng/g gentamicin limit of the FAST applied to the fresh kidney of the recently slaughtered bovine.

LC/MS/MS measurement of penicillin G in bovine plasma, urine, and biopsy samples taken from kidneys of standing animals.

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals.

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.

Cytological and microbiological findings in guttural pouch lavages of clinically normal horses with head restraint.

OBJECTIVE: To evaluate the cytological and microbiological contents of guttural pouch washes of ten randomly selected horses restrained so as to prevent them lowering their heads, and to assess the possible effects on the guttural pouch environment in these horses. PROCEDURE: Cytological and microbiological studies were performed on guttural pouch washes of ten clinically normal horses restrained in a standing position so as to prevent them from lowering their heads below normal, as would happen during transportation on long journeys. They were restrained for 12 or 24 h and cytological, bacteriological and mycological findings in guttural pouch washes were recorded. RESULTS: The cytological gradings and neutrophil concentrations of guttural pouch washings were higher in horses that had their heads restrained for a longer period. Washings from these horses were more likely to contain cultivable bacteria and were the only washes yielding potentially pathogenic bacterial species. CONCLUSION: Variation in the cytological differential counts and bacterial cultures of guttural pouch lavages may be found in clinically normal horses which have had their heads restrained in an elevated position for periods from 12 to 24 h. This should be considered when examining this site and care must be taken when interpreting cytology of guttural pouch lavages in samples taken after transportation for more than 12 h. Restriction of head movement could also affect the normal pouch enviroment and predispose it to disease.