Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis.

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l-methadone and d-methadone and their major metabolites, l- and d-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d-methadone concentrations were lower than those of l-methadone and the d-EDDP levels were lower than those of L-EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two-compartment pharmacokinetic model. l-methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady-state volume of distribution than d-methadone. d-methadone and d-EDDP were eliminated faster than their respective l-enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.

Combined caudal retrocostal and lateral ultrasound-guided approach for transversus abdominis plane injection: A descriptive pilot study in pig cadavers.

Transversus abdominis plane (TAP) block is a regional anesthetic technique used to desensitize the abdominal wall in several species. This study aimed to describe the anatomical characteristics of the abdominal wall and to identify a feasible approach for an US-guided TAP injection that would result in adequate staining of the relevant nerves in the abdominal wall in pig cadavers. Fresh cadavers from five Landrace pigs (age, 12 weeks; body weight, 35.5 ± 1.6 kg) were used. One pig (n = 1) was anatomically dissected, and four pigs (n = 4; i.e., 8 hemiabdomens) were used for TAP injections and evaluation of dye spread. The volume of 0.3 mL/kg/injection point of methylene blue was injected bilaterally. In the caudal retrocostal approach, the injection was performed ventral to the most caudal part of the costal arch. In the lateral approach, the injection was performed between the last rib and iliac crest. A needle was inserted in plane for the caudal retrocostal and the lateral approach caudocranially and craniocaudally, respectively. Successful staining was defined as presence of dye on the nerve for a length of >1 cm in its entire circumference. The TAP was found between different muscle layers in the described anatomical regions. In the caudal retrocostal approach the TAP was found between the external abdominal oblique and transversus abdominis muscle bellies. In the lateral approach the TAP was found between the internal abdominal oblique and transversus abdominis muscles. The approach combining lateral and caudal retrocostal injections at the studied volume stained a median of 5 (3-6) target nerves from the fourth-last thoracic nerve to L2 (six nerves). Combined caudal retrocostal and lateral TAP injections of 0.3 mL/kg/injection point, resulted in staining of target nerve branches which supply the periumbilical and caudal abdominal wall in pig cadavers.

Pharmacokinetics and pharmacodynamics of l-methadone in isoflurane-anaesthetized and mechanically ventilated ponies.

OBJECTIVE: To evaluate the pharmacokinetics and selected pharmacodynamic effects of a commercially available l-methadone/fenpipramide combination administered to isoflurane anaesthetized ponies. STUDY DESIGN: Prospective single-group interventional study. ANIMALS: A group of six healthy adult research ponies (four mares, two geldings). METHODS: Ponies were sedated with intravenous (IV) detomidine (0.02 mg kg-1) and butorphanol (0.01 mg kg-1) for an unrelated study. Additional IV detomidine (0.004 mg kg-1) was administered 85 minutes later, followed by induction of anaesthesia using IV diazepam (0.05 mg kg-1) and ketamine (2.2 mg kg-1). Anaesthesia was maintained with isoflurane in oxygen. Baseline readings were taken after 15 minutes of stable isoflurane anaesthesia. l-Methadone (0.25 mg kg-1) with fenpipramide (0.0125 mg kg-1) was then administered IV. Selected cardiorespiratory variables were recorded every 10 minutes and compared to baseline using the Wilcoxon signed-rank test. Adverse events were recorded. Arterial plasma samples for analysis of plasma concentrations and pharmacokinetics of l-methadone were collected throughout anaesthesia at predetermined time points. Data are shown as mean ± standard deviation or median and interquartile range (p < 0.05). RESULTS: Plasma concentrations of l-methadone showed a rapid initial distribution phase followed by a slower elimination phase which is best described with a two-compartment model. The terminal half-life was 44.3 ± 18.0 minutes, volume of distribution 0.43 ± 0.12 L kg-1 and plasma clearance 7.77 ± 1.98 mL minute-1 kg-1. Mean arterial blood pressure increased from 85 (±16) at baseline to 100 (±26) 10 minutes after l-methadone/fenpipramide administration (p = 0.031). Heart rate remained constant. In two ponies fasciculations occurred at different time points after l-methadone administration. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of a l-methadone/fenpipramide combination to isoflurane anaesthetized ponies led to a transient increase in blood pressure without concurrent increases in heart rate. Pharmacokinetics of l-methadone were similar to those reported for conscious horses administered racemic methadone.

Ultrasound-Guided Transversus Abdominis Plane Block in Shetland Ponies: A Description of a Three-Point Injection Technique and Evaluation of Potential Analgesic Effects.

Colic surgery is one of the most painful procedures carried out in horses. Common strategies to alleviate immediate postsurgical abdominal pain include the administration of potent systemic analgesics; however, these may cause unwanted adverse effects such as cardiovascular depression, ileus, and ataxia. The administration of local anesthetics at the incision site in form of an ultrasound-guided subcostal transversus abdominis plane (TAP) block may therefore be preferred to provide adequate analgesia without significant side effects. To date, no technique for a TAP block in horses undergoing median celiotomy has been described. The objective of the study was to develop a TAP block technique, which will lead to the desensitization of the ventrolateral abdominal wall and adjacent skin area of experimental Shetland Ponies using bupivacaine 0.125%. This is a prospective, blinded, self-controlled trial. A cadaver study was performed to determine the ideal injection points and the volume required to stain the nerves responsible for the sensation of the ventrolateral abdominal wall and skin in Shetland pony cadavers (i.e., T9-L 2). Subsequently, using the ideal injectate volume and the landmarks obtained in the first phase of the study, six Shetland ponies received a bilateral TAP injection, either with a local anesthetic solution (bupivacaine 0.125%) or with saline in a randomized, crossover, blinded fashion. Effectiveness was determined over a 4 hour postinjection time, by using a pinprick technique. Significant differences were found to the responses of the pinprick evaluation between the bupivacaine- and saline-treated sides after 30 minutes of TAP block injection. Reported "learned behavior" could have affected the results of the pinprick testing. The TAP block technique reported in this study using bupivacaine 0.125% appeared effective in desensitizing the lower abdomen of ponies for up to 2 hours. Further research is required to apply this technique in horses undergoing celiotomy. Potentially larger volumes and/or higher concentrations of bupivacaine may be necessary to provide longer duration of action of the block.

Use of a colorimetric assay to evaluate the proliferation of canine mammary tumor cells exposed to propofol.

Drugs applied on human cancer cells can influence the rate of cell proliferation. The present study investigates the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) colorimetric assay to evaluate canine tumor cell proliferation after exposure to the injectable anesthetic, propofol. Primary (CIPp) and metastatic (CIPm) canine tubular adenocarcinoma cell lines were incubated with cell culture medium (control) or propofol (1, 5, and 10 µg/mL). The MTT assays were performed after 6 and 12 hours of exposure. Measurements of absorbance were obtained for each condition with a spectrophotometer and compared with controls using a 3-way analysis of variance (P < 0.05). An increased cell proliferation rate was observed in CIPp exposed to 5 and 10 µg/mL of propofol for 6 hours and 1, 5, and 10 µg/mL for 12 hours. No significant changes were observed in CIPm after 6 hours of exposure. All propofol concentrations decreased the cell proliferation rate in CIPm after 12 hours of exposure. The MTT assays showed that exposure of CIPp to propofol for 6 and 12 hours increased cell proliferation. A decrease in the CIPm proliferation rate was observed when propofol exposure lasted for 12 hours. Further studies are warranted to better understand the role of propofol on cancer cell proliferation.

Les médicaments appliqués sur les cellules cancéreuses humaines peuvent influencer la vitesse de prolifération cellulaire. La présente étude a examiné l'utilisation du test colorimétrique au bromure de 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tétrasodique (MTT) pour évaluer la prolifération de cellules tumorales canines après exposition à l'anesthésique injectable, propofol.Des lignées cellulaires primaires (CIPp) et métastasiques (CIPm) d'adénocarcinome tubulaire canin furent incubées avec du milieu de culture cellulaire (témoin) ou du propofol (1, 5, et 10 µg/mL). Les tests au MTT ont été effectués après 6 et 12 h d'exposition. Les mesures d'absorbance furent obtenues pour chaque condition à l'aide d'un spectrophotomètre et comparées aux témoins en utilisant une analyse de variance à trois facteurs (P < 0,05).Une augmentation de la vitesse de prolifération cellulaire fut observée chez les CIPp exposées à 5 et 10 µg/mL de propofol pour 6 h et à 1, 5, et 10 µg/mL pour 12 h. Aucun changement significatif ne fut observé chez les CIPm après 6 h d'exposition. Toutes les concentrations de propofol ont réduit la vitesse de prolifération cellulaire des CIPm après 12 h d'exposition.Les tests au MTT ont démontré que l'exposition de CIPp au propofol pour 6 et 12 h augmentait la prolifération cellulaire. Une réduction de la vitesse de prolifération des CIPm fut observée lorsque l'exposition au propofol durait 12 h. Des études supplémentaires sont nécessaires pour mieux comprendre le rôle du propofol sur la prolifération des cellules cancéreuses.(Traduit par Docteur Serge Messier).

Canine mammary tumour cells exposure to sevoflurane: effects on cell proliferation and neuroepithelial transforming gene 1 expression.

OBJECTIVE: The influence of perioperative factors, such as anaesthetic and analgesic techniques, on metastatic spread following surgery for primary cancer removal is of growing interest. The present study investigated the effects of sevoflurane on canine mammary tumour cell proliferation (MTT colorimetric assay) and on the expression of neuroepithelial transforming gene 1 (NET1). STUDY DESIGN: Prospective controlled in vitro trial. STUDY MATERIAL: Primary (CIPp) and metastatic canine tubular adenocarcinoma (CIPm) cells. METHODS: To perform MTT tests, cell lines were seeded at a density of 3000 cells per well and incubated with sevoflurane (1, 2.5 or 4 mM) or only with the culture medium (control). Sevoflurane was added to the cell cultures every hour to avoid changes in drug concentration. MTT assays were performed after 6 hours of exposure obtaining absolute values of absorbance. The RNA isolated from the lysates of the same cell lines underwent quantitative polymerase chain reaction to evaluate NET1 gene expression changes compared with controls. One- or two-way analysis of variance was used as appropriate (p < 0.05). RESULTS: A significant increase in cell proliferation compared with controls was observed in CIPp treated with lower sevoflurane concentrations, whereas a significant decrease in cell proliferation was found in CIPm treated with all the sevoflurane concentrations. All CIPp treatments did not induce changes in gene expression compared with controls, whereas a significant increase in gene expression was observed in CIPm between controls and the higher sevoflurane concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Sevoflurane treatments modified the cell proliferation rate in both cell lines showing an increase or decrease when applied to CIPp or CIPm, respectively. Expression of the NET1 gene increased after treatment with sevoflurane 4 mM in metastatic cells. The role of sevoflurane on cancer recurrence should be further investigated.

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane.

An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ-cyclodextrin. The developed assay is precise (intra- and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.

Femoral and sciatic nerve blockades and incision site infiltration in rabbits undergoing stifle joint arthrotomy.

This study was designed to determine whether perineural injections of local anaesthetics decreases intraoperative nociception and improves postoperative analgesia in New Zealand White rabbits undergoing experimental stifle arthrotomy. All animals were anaesthetized using isoflurane and received morphine intramuscularly. The sciatic and femoral nerves of the leg to be operated on were located using a nerve stimulator (1 Hz, 0.5 mA). Rabbits were assigned to a treatment group (LB; n = 12) or a placebo group (P; n = 12) in a randomized blinded fashion. Group LB received lidocaine 2% (1 mg/kg) combined with bupivacaine 0.5% (0.25 mg/kg) injections around the sciatic and femoral nerves (0.1 mL/kg total volume per site) and subcutaneous infiltration of the incision site with lidocaine 1% (1.25 mg/kg). Group P received the same volume of 0.9% NaCl. Rabbits in group P required higher doses of intraoperative fentanyl and propofol to reduce heart rate and suppress increase in systolic blood pressure, and maintain an adequate anaesthetic plane. Interventional analgesia (buprenorphine and carprofen) was required significantly earlier in rabbits in group P (2 and 6 h after the first nerve blockade, respectively) based on assessment of their pain-related behaviour and range of motion. Using a visual analogue scale (0 mm= no pain to 100 mm= maximal possible pain), rabbits in group LB were judged to show significantly less pain compared with rabbits in group P (14 ± 10 mm and 37 ± 25 mm, respectively) 2 h after nerve blockade. In conclusion, this technique of perineural analgesia combined with incision site infiltration reduced intraoperative fentanyl requirements and improved postoperative analgesia in rabbits.

Cardiopulmonary effects and anaesthesia recovery quality in horses anaesthetized with isoflurane and low-dose S-ketamine or medetomidine infusions.

OBJECTIVES: To evaluate cardiopulmonary effects and anaesthesia recovery quality in horses anaesthetized with isoflurane receiving medetomidine or S-ketamine infusions. STUDY DESIGN: Randomized, blinded, prospective clinical trial. ANIMALS: Fifty horses undergoing elective surgery. METHODS: After acepromazine and flunixin meglumine premedication, horses received medetomidine (7 µg kg-1 ) intravenously (IV). Anaesthesia was induced with midazolam and racemic ketamine (Med treatment group; 2.2 mg kg-1 ; n = 25) or S-ketamine (S-ket treatment group; 1.1 mg kg-1 ; n = 25) IV and maintained with isoflurane in oxygen/air and medetomidine (Med; 3.5 µg kg-1 hour-1 ) or S-ketamine (S-ket; 0.5 mg kg-1 hour-1 ). All horses were mechanically ventilated. Cardiopulmonary variables were evaluated. Isoflurane end-tidal concentrations (Fe'Iso), dobutamine requirements and thiopental boli were recorded. Plasma samples were collected in six horses to evaluate S-ketamine and S-norketamine concentrations. After surgery, medetomidine 2 µg kg-1 was administered IV. Four independent observers scored recovery using a visual analogue scale and a numerical rating scale. RESULTS: Both groups required similar mean Fe'Iso (1%). However, S-ket horses needed more thiopental boli. Median intraoperative cardiac index values were higher with S-ket (4.5 L minute-1  m-2 ) than Med (3.9 L minute-1  m-2 ). Overall, there were no differences in heart rate, blood pressure or dobutamine requirements; however, horses in S-ket showed higher heart rate values at 30 minutes after anaesthesia induction. Compared with Med horses, S-ket horses showed decreased PaO2 and increased pulmonary venous admixture values estimated with the Fshunt calculation. Recoveries were shorter and of poorer quality with S-ket. During infusion, S-ketamine and S-norketamine plasma concentrations lay in the ranges of 0.209-0.917 µg mL-1 and 0.250-0.723 µg mL-1 , respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the higher intraoperative cardiac index with S-ket, both protocols were considered to provide acceptable cardiovascular function. However, recovery quality was significantly better in the Med group.

Influence of anesthetic variables on short-term and overall survival rates in cats undergoing renal transplantation surgery.

OBJECTIVE: To identify factors associated with short-term (30-day) and overall survival rates in cats that underwent renal transplantation surgery (RTS). DESIGN: Retrospective cohort study. ANIMALS: 94 cats that underwent RTS from 1998 through 2010. PROCEDURES: Data obtained from the medical records pertinent to RTS included cat signalment; anesthetic agents, techniques, and timings; supportive treatment; perioperative physiologic findings; and surgery and warm ischemia times. Associations with short-term and overall survival rates were investigated. RESULTS: Median survival time was 653 days (range, 2 to 4,580 days). Prolonged anesthesia (median, 300 minutes; range, 225 to 445 minutes) reduced overall survival rate but did not influence short-term survival rate. No associations were identified between survival rates and anesthetic agent used, amount and type of fluid administered IV, physiologic abnormalities, and blood product administration. All cats that received µ-opioid receptor antagonists at anesthetic recovery to reverse the effects of µ-opioid receptor agonists survived for at least 30 days. High Hct at the end of anesthesia was also associated with an increase in short-term survival rate. Two cats had an intraoperative hemoglobin oxygen saturation < 90%, and both died within 7 days after surgery. Cats > 12 years old had a lower overall survival rate than did younger cats. CONCLUSIONS AND CLINICAL RELEVANCE: Minimization of total anesthesia time, reversal of µ-opioid receptor agonists at the end of anesthesia, and prevention of intraoperative decreases in blood oxygen saturation and postoperative decreases in Hct appeared to help maximize postsurgical survival time in cats undergoing RTS.

Comparison of caudal and pre-scrotal castration for management of perineal hernia in dogs between 2004 and 2014.

AIMS: To compare peri- and post-operative complications associated with caudal scrotal castration (CSC) and perineal hernia repair with pre-scrotal castration (PSC) in conjunction with another surgical procedure. METHODS: Medical records were reviewed for 51 intact male dogs that were admitted to the Veterinary Emergency and Surgical Center, Levittown, PA, and underwent a CSC and perineal hernia repair using an internal obturator muscle flap (IOMF) between 2004 and 2014. Perioperative, and major and minor post-operative complications noted within the 2 week follow up period were reported and compared to 91 intact male dogs that underwent a PSC in conjunction with a second surgical procedure. RESULTS: There were no recorded perioperative or major post-operative complications in either group. There were 3/51 (6%) minor post-operative complications in the CSC group compared to 6/91 (7%) in the PSC group. There were 2/51 (4%) and 4/91 (4%) cases that developed heat, erythema and swelling associated with the incision site and 1/51 (2%) and 2/91 (2%) cases that developed scrotal swelling in the CSC and PSC groups, respectively. Overall, there was no difference in the prevalence of minor complications between the two groups (p=0.86). CONCLUSIONS AND CLINICAL RELEVANCE: Caudal scrotal castration was not associated with more perioperative or postoperative complications relative to PSC. Utilising the CSC approach eliminates the need to aseptically prepare and drape a second site when carrying out perineal hernia repair, as well as the need for patient repositioning. Thus, we recommend that CSC be the preferred surgical technique when performing orchiectomy in dogs concurrent with perineal hernia repair.

Trending ability and limitations of transpulmonary thermodilution and pulse contour cardiac output measurement in cats as a model for pediatric patients.

The present study evaluated transpulmonary thermodilution (TPTD) and pulse contour cardiac output (PCCO) both measured by the PiCCO Plus™ monitor (Pulsion Medical Systems, Munich, Germany) against pulmonary artery thermodilution (PATD) in cats as a hemodynamic model for small children. A wide range of cardiac outputs (CO) was simultaneously measured. Accuracy and trending abilities were critically evaluated. Three cats were studied under isoflurane anesthesia and 160 CO measurements were performed with 3 mL ice-cold 5 % dextrose with PATD and TPTD. The results were compared with the PCCO measurement before the bolus measurement. Cardiac output was manipulated from 32 to 224 mL/kg/min by dobutamine, dopamine, phenylephrine, medetomidine and increased concentrations of isoflurane. Bland-Altman analysis, concordance and polar plot analysis were performed to assess accuracy and trending ability. TPTD was measuring constantly higher than PATD with a mean bias of 73 mL/kg/min and limits of agreement of 34-112 mL/kg/min, a concordance rate of 94 % and a mean polar angle of -5° with radial limits of agreement (RLOA) of 33°. Concordance rate of the PCCO versus PATD was 82 % with a mean polar angle of -10° and RLOA of 46° and versus TPTD 90 % with a mean polar angle of -6° and RLOA of 46°. Both tested methods constantly overestimated simultaneous PATD measurements. The small size, low flows and the relative short catheter not reaching the abdominal aorta may explain that. However TPTD tracked changes accurately opposed to a poor trending ability of the PCCO measurement.

Effects of acepromazine or methadone on midazolam-induced behavioral reactions in dogs.

This study evaluated whether acepromazine or methadone reduced behavioral parameters, overall excitement, and activity associated with midazolam administration to healthy dogs. Dogs received midazolam (M) alone [M: 0.25 mg/kg body weight (BW)] or with methadone (MM) (MM: 0.75 mg/kg BW) or acepromazine (MA) (MA: 0.03 mg/kg BW) or saline (S) solution alone, all intramuscularly. Two blinded observers evaluated behavioral parameters using video recordings 30 min before and after injection of drugs. Accelerometery was used to evaluate "total activity counts" (TAC) at baseline and post-treatment. Post-treatment excitement scores were significantly higher in M and MA compared to baseline, M and MM compared to S, and M compared to MA. Behavioral parameters showed significantly higher proportions of "pacing" post-treatment in all groups receiving midazolam, and "restlessness," "chewing/licking," and "sniffing" in M. No significant differences were found for TAC at baseline and post-treatment. Midazolam-induced paradoxical behavioral changes (excitation, panting, pacing, restlessness, licking/chewing, and vocalization) were not prevented by acepromazine or methadone in healthy dogs.

Effets de l'acépromazine ou de la méthadone sur les réactions comportementales induites par le midazolam chez les chiens. Cette étude a évalué si l'acépromazine ou la méthadone réduisait les paramètres comportementaux, le niveau d'excitation général et l'activité associée à l'administration de midazolam chez des chiens en santé. Les chiens ont reçu le midazolam (M) seul (M : 0,25 mg/kg poids corporel [PC]) ou avec de la méthadone (MM) (MM : 0,75 mg/kg PC) ou de l'acépromazine (MA) (MA : 0,03 mg/kg PC) ou une solution saline (S) seule, tous administrés par voie intramusculaire. Deux observateurs à l'aveugle ont évalué les paramètres comportementaux à l'aide d'enregistrements vidéo 30 minutes avant et après l'injection des médicaments. Un accéléromètre a été utilisé pour évaluer les «numérations de l'activité totale¼ (NAT) comme données de référence et après le traitement. Les notes d'excitation après le traitement étaient significativement supérieures pour M et MA comparativement aux données de référence, M et MM comparativement à S et M comparativement à MA. Les paramètres comportementaux ont montré des proportions significativement supérieures de «va-et-vient¼ après le traitement dans tous les groupes qui avaient reçu midazolam et une «agitation¼, de «mastication et léchage¼ et de «reniflement¼ dans M. Aucune différence significative n'a été constatée pour NAT aux données de référence et après le traitement. Les changements comportementaux paradoxes induits par le midazolam (excitation, halètement, va-et-vient, agitation, lèchage et mastication et vocalisation) n'ont pas été prévenus par l'acépromazine ni la méthadone chez les chiens en santé.(Traduit par Isabelle Vallières).

Nematode challenge induces differential expression of oxidant, antioxidant and mucous genes down the longitudinal axis of the sheep gut.

To characterize the role of a range of oxidant, antioxidant and mucous-related genes in the primary response to gastrointestinal nematodes, groups of genetically resistant sheep were challenged with either Haemonchus contortus or Trichostrongylus colubriformis and necropsied for retrieval of tissue at days 0, 3, 7, 14 and 21. To determine if the response was localized to the site of parasite infection, four different gut tissues were sampled: the abomasum, proximal and distal jejunum and ileum. Basal expression patterns of all candidate genes were determined using the day 0 (pre-challenge) samples. A conserved innate response involving elevated expression of dual oxidase, glutathione peroxidase and trefoil factor was initiated within 3 days of challenge and extended out to 21 days. An increase in host gene expression levels at the preferred site of infection (the abomasum for H. contortus and the proximal jejunum for T. colubriformis) was also common to both nematodes. However, these increases were concomitant with reduced expression in other areas of the gut suggesting a compartmentalized response. Other aspects of the response were parasite-specific, with T. colubriformis challenge inducing expression peaks at times corresponding to nematode life-stage transitions.

Sequence diversity and rates of molecular evolution between sheep and cattle genes.

Experiments that aim to identify genes of importance in sheep are currently inhibited by a paucity of genomic resources. One approach, therefore, is to exploit the wealth of data and associated capabilities becoming available for the bovine genome. Cross-species application of microarrays and comparative sequencing to identify single nucleotide polymorphisms are two possibilities; however, both are dependant on the level of nucleotide sequence similarity between the two species. This study used 120 gene orthologues consisting of over 60 kb of aligned sequence to estimate the gene diversity between cattle and sheep. Less than 3% of protein-coding nucleotide positions were found to be different, indicating that the prospect for successfully using cross-species strategies is high. Substitution at synonymous sites ranged between 6.9 and 7.7% (+/- 0.3%), and was higher than at non-synonymous sites (1.4-1.7 +/- 0.1%). The relative rate test was used to determine whether the observed mutation rates were constant between the two lineages. While the rate at synonymous sites appeared constant, the rate at non-synonymous sites was significantly higher within the caprinae lineage (sheep) when compared with bovinae (cattle; chi2 = 10.03; d.f. = 1, P < 0.01). This is the first demonstration that variable rates of molecular evolution may be present within the family Bovidae.

Potential for recombinant Babesia bovis antigens to protect against a highly virulent isolate.

Two antigens from Babesia bovis,12D3 and 11C5, were expressed and purified as recombinant proteins in Escherichia coli and used to vaccinate groups of six Babesia-susceptible cattle. These were subsequently challenged with a highly virulent strain of B. bovis. All cattle showed symptoms of disease and most required treatment. Cattle vaccination groups receiving either 12D3 or 11C5 or a combination of both, reduced parasitaemia by approximately fourfold and a number of individual animals appeared to control the parasite infection. Control of parasites correlated with high monocyte numbers late in infection. The results thus confirm the potential usefulness of both antigens but also demonstrate the limitations of current formulations.

Development of a mathematical method for classifying and comparing tree architecture using parameters from a topological model of a trifurcating botanical tree.

This paper describes a model for the topological mapping of trifurcating botanical trees. The model was based on a system of modular units that represented the interconnectivity of shoot meristems (terminal segments) and internodes (internal segments) within whole plant canopies, organized with increasing centrifugal ordering. The model was capable of describing the dynamics of plant growth as expressed by changes in topological parameters over time. Preliminary calculations for experimental trees indicated that the model represents growth in a biologically sound manner. Methods are described for the calculation of the architecture parameters size, size-complexity, structural complexity, and tree asymmetry index (TAI). Parameter calculations were based on the mathematical principles developed for the classification of bifurcating dendrite trees, and were designed to both extract structural information, and to enable statistical comparison between trees of different size. Parameters were mathematically adjusted for trifurcation, and appeared to be able to represent quantitatively the architectural properties of tree structures. In addition to the calculation of the TAI for trifurcating trees, new methods were developed to enable comparisons to be made of the architectural complexity of trifurcating trees of differing size. These were based on the principle of the pair-wise comparison of the mean centrifugal order number (MCON) with respect to segments against highest order number. We argue and illustrate that this principle can be more informative than that of pair-wise comparison of the MCON against tree degree (topological size). Further improvements to this method were made by examining branching points (vertices) rather than segments (links) to calculate the MCON.

Generation of a transcription map distal to HLA-F.

We have constructed a transcription map covering a 2 Mb region beginning approximately 1 Mb distal to HLA-F. Cosmids isolated from a chromsome 6 library were positioned by YAC hybridisation, STS and fingerprint analysis. Using direct cDNA selection, exon trapping, and direct genomic sequence analysis, we identified 42 potential exonic fragments in this region. Six fragments corresponded to previously characterised genes, four previously broadly mapped to this region. Five fragments were similar to known genes, eight fragments matched ESTs and 10 of the remaining 23 novel fragments, gave a positive signal on northern analysis. All cDNA fragments were mapped to the YAC and cosmid contig covering the region and with respect to other known genes and STS in this area. The distribution of the cDNA fragments indicated their organisation in three clusters around CpG islands.

Identification of a novel Krueppel-related zinc finger gene (ZNF184) mapping to 6p21.3.

cDNA selection and exon trapping were performed on cosmids mapping to a region 3 Mb distal to HLA-A. Analysis of resulting fragments indicated the presence of two zinc finger transcripts, and one of these was used to isolate a partial cDNA (ZNF184) from a placental library. The second transcript contained additional sequence of the 5' end of the gene, extending the sequence to 2678 bp. Sequence analysis indicates that ZNF184 is a classical Krueppel zinc finger with 19 highly conserved zinc finger motifs at the C-terminus and a Krueppel associated box at the N-terminus of the protein. This gene encodes a 3.2-kb transcript that is highly expressed in testis and expressed at a moderate to low level in all other tissues tested. This zinc finger gene maps to a region approximately 200 kb distal to the microsatellite marker D6S105 and approximately 300 kb proximal to D6S1260.