Animal-based measurements of the severity of mastitis in dairy cows.

A range of clinical parameters were studied to assess their usefulness as objective markers of the severity of clinical mastitis in dairy cows. Cows with moderate clinical mastitis had significantly higher rectal temperatures and heart and respiratory rates than cows with mild clinical mastitis or control cows. The difference in temperature between quarters did not vary significantly between the control cows and the cows with mastitis, but there was a larger difference between quarter temperatures in the cows with moderate mastitis than in the cows with mild mastitis or the control cows. The hock-to-hock distance in the control cows was significantly smaller than in the cows with mild to moderate mastitis, but there was no significant difference in the distance either between the cows with mild mastitis and those with moderate mastitis, or between the cows with mastitis in the front quarters and those with mastitis in the hind quarters. The mechanical threshold to pain of the cows with mild and moderate mastitis was significantly lower than that of the control cows.

Measuring pain in dogs and cats using structured behavioural observation.

The contemporary approach to pain measurement in people and animals seeks to measure the affective (emotional) component of the pain experience using structured questionnaires with formal scoring methodology. Chronic pain has wide-ranging impacts which affects the quality of life (QOL) of the individual, whether that is a person or an animal. Accordingly instruments to measure chronic pain are designed to measure its impact on QOL and are called health-related quality of life (HRQL) instruments. In veterinary science instruments to measure pain are based on behavioural observation by the veterinary surgeon/nurse in the case of acute pain and by the owner in the case of chronic pain. The development of HRQL instruments is an expanding field in veterinary science, not just for the measurement of pain, but for other chronic diseases, and it has a wide application in pharmaceutical research and clinical practice to improve patient care. This review highlights the challenges involved in creating such measures for dogs and cats, seeking to provide the reader with an understanding of their development process. It then provides an overview of the current status with regard to acute and chronic pain measurement.

Pain assessment following experimental maxillofacial surgical procedure in sheep.

The aim of this study was to investigate the severity and duration of postoperative pain and hyperalgesia in sheep undergoing mandibular reconstructive surgery. Stimulus-evoked sensitivity at the surgical site and an area remote from injury, the ipsilateral and contralateral forelimbs, was measured as objective indicators of altered pain processing in adult female sheep (n = 7). Responses were recorded before surgery and one, two, three, seven and 14 days afterwards. Concentrations of the acute-phase protein haptoglobin were measured in serum as a marker of inflammation before and at one and seven days after surgery. A significant decrease in forelimb mechanical withdrawal thresholds (secondary hyperalgesia) and response thresholds to punctate stimulation of the area surrounding the surgical incision (allodynia) was detected one day after surgery and persisted for at least three days, despite intra- and postoperative analgesic treatment. Concentrations of haptoglobin were significantly increased one day post-surgery, indicating the presence of a significant acute inflammatory response, and returned to pre-surgical concentrations by seven days. These data provide a deeper insight into understanding the impact of surgery in experimental animals, and may assist in formulating more effective analgesic and antihyperalgesic treatment regimens postoperatively.

Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis.

The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M-SAA3 in mammary tissues was determined using immunocytochemistry. Increases of M-SAA3 and Hp in milk occurred within 12 h of Staphylococcus aureus infusion, with peak concentrations occurring 3 d after infusion of the bacteria. The increase of acute phase proteins in milk (15 h) preceded the increase in serum concentrations of both proteins (24 h). Expression of mRNA for M-SAA3 and Hp increased in both mammary and hepatic tissues 48 h after infusion of the mammary glands. In mammary tissue, the increase of M-SAA3 mRNA was greater than the increase in Hp mRNA expression, whereas in hepatic tissue, the increase in M-SAA3 mRNA was less than that for Hp mRNA. Immunocytochemistry demonstrated that M-SAA3 protein was present within secretory epithelial cells at significantly higher levels in infected mammary glands than in control tissues. These proteins, which have host defense and antibacterial activities, may play a significant role in the early response to invasion of mammary tissues by pathogenic bacteria.

Behavioural evidence supporting a differential role for group I and II metabotropic glutamate receptors in spinal nociceptive transmission.

Metabotropic glutamate receptors (mGluRs) have been shown to contribute to nociceptive processing in spinal cord. This study examined the effects of intrathecal treatment with group I and II mGluR compounds on withdrawal thresholds to noxious mechanical stimuli, in the absence of tissue damage or inflammation, in adult female sheep. Both the group I/II mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 5.2-520 nmol) and the group II agonist (2S,1S, 2S)-2-(carboxycyclopropyl)glycine (L-CCG-I; 620 nmol) significantly increased mechanical withdrawal thresholds between 5-15 min post-injection. These anti-nociceptive effects were blocked by co-administration of the mGluR antagonist (2S)-alpha-ethylglutamate (EGLU; 570 nmol; group II), but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 450 nmol; group I). Intrathecal administration of the group I-specific agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG; 50 nmol) produced a significant reduction in mechanical thresholds, which was blocked by co-administration of the group I antagonist AIDA. In contrast, the highest dose of (S)-3,5-DHPG tested, 5 micromol, significantly elevated response thresholds. These results demonstrate that both group I and II mGluRs play crucial, but contrasting roles in mediating acute mechanical nociceptive events in spinal cord.

Behavioral evidence supporting a differential role for spinal group I and II metabotropic glutamate receptors in inflammatory hyperalgesia in sheep.

A differential role for metabotropic glutamate receptors (mGluRs) in spinal nociception in normal animals has previously been identified. The present study examined the contribution of group I and group II mGluRs to the development and maintenance of inflammatory hyperalgesia produced by unilateral intradermal injection of carrageenan into the lower forelimb in sheep. Carrageenan (7.5 mg in 500 micro l) produced a significant bilateral reduction in forelimb mechanical withdrawal thresholds. Intrathecal administration of saline-vehicle or the group II mGluR antagonist (2S)-alpha-ethylglutamate (EGLU; 570 nmol) had no effect on either the development or maintenance of hyperalgesia. However, intrathecal administration of the group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 450 nmol) before carrageenan blocked the development of ipsilateral hyperalgesia, and when given 2 h after carrageenan, reversed both ipsilateral and contralateral hyperalgesia. Intrathecal administration of the group II mGluR agonist (2S,1S,2S)-2-(carboxycyclopropyl)glycine (L-CCG-I; 620 nmol) given either before or after carrageenan treatment produced analgesia and anti-hyperalgesia, an effect abolished by co-administration of EGLU (570 nmol). The magnitude of the analgesic response, assessed by the area under the response curve, was significantly greater than that produced by LCCG-I in normal animals. These data demonstrate that the development and maintenance of inflammatory hyperalgesia is dependent on activation of group I mGluRs in spinal cord. In addition, the analgesic and anti-hyperalgesic actions of group II mGluRs suggest that these receptors play a crucial role in modulating acute inflammatory hyperalgesia.

Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release.

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.

The role of nitric oxide in the responses of the ovine digital artery to vasoactive agents and modification of these responses by endotoxin and cytokines.

1. Laminitis, an important cause of lameness in domestic ungulates, occurs as a result of altered digital perfusion. Endotoxin and cytokines may mediate the vascular derangements observed through alterations in nitric oxide production. In this study, the vascular responses of the isolated ovine digital artery were examined and the influence of endotoxin and cytokines investigated. 2. Neither removal of the endothelium nor incubation with N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) altered the response to phenylephrine (PE, 1 nM to 300 microM). Indomethacin (10 microM) decreased PE log EC(50) from -6.22+/-0.08 to -6.55+/-0.07. Acetylcholine (1 nM to 1 mM) and bradykinin (BK, 100 pM to 3 microM) induced endothelium-dependent relaxation. Bradykinin-induced relaxation was reduced by L-NAME, E(max) falling from -61.7+/-7.4 to -34.0+/-2.1%. Addition of indomethacin further reduced BK E(max) to -9.6+/-2.8%. Sodium nitroprusside (1 nM to 300 microM) produced endothelium-independent relaxation that was unaffected by L-NAME or indomethacin. 3. Following a 6 h incubation with endotoxin (3 microml(-1)), arterial responses to PE and BK did not differ from polymyxin B-treated controls (10 microg ml(-1)). Arteries incubated for 6 h with interferon-gamma (IFN-gamma, 10 ng ml(-1)) and tumour necrosis factor-alpha (TNF-alpha, 5 ng ml(-1)) exhibited greater relaxation to BK (E(max)-50.0+/-5.1%) than polymyxin B-treated controls (E(max)-33.1+/-4.0%), but did not differ in their response to PE. 4. Prolonged incubation (16 h) with endotoxin (3 microg ml(-1)) did not alter the response to PE, however incubation with IFN-gamma (10 ng ml(-1)), TNF-alpha (5 ng ml(-1)) and interleukin-1beta (20 ng ml(-1)) for 16 h increased PE log EC(50) from -6.44+/-0.09 to -6. 10+/-0.11. 5. Nitric oxide is an important mediator of endothelium-dependent relaxation in ovine digital arteries but does not modulate PE-induced vasoconstriction. Incubation with cytokines decreased the sensitivity of digital arteries to PE.

N-methyl D-aspartate induced mechanical allodynia is blocked by nitric oxide synthase and cyclooxygenase-2 inhibitors.

The role of spinal NMDA receptors in mechanical nociceptive processing was assessed in sheep. Intrathecal NMDA (2 nmol-1 micromol) produced a significant reduction in mechanical withdrawal thresholds. This effect was attenuated by pretreatment with the NMDA receptor antagonist MK801 (100 nmol), the cyclooxygenase-2 (COX-2) inhibitor 5,5-dimethyl-3-(3-flourophenyl)-4-(4-methylsulphonyl)phenyl-2(5H) furanone DFU; 200 nmol) and the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 2 micromol), but not by the metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 200 nmol-2 micromol) or the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 200 nmol-1 micromol). This first report of NMDA-induced mechanical allodynia suggests that spinal NMDA receptors are involved in mediating acute mechanical nociceptive processing through activation of NOS and COX-2 enzymes.

Biphasic modulation of nociceptive processing by the cyclic AMP-protein kinase A signalling pathway in sheep spinal cord.

A role for the cyclic AMP (cAMP)-protein kinase A (PKA) transduction cascade in nociceptive processing has been identified. This study examined the effects of intrathecal treatment with the cAMP analogue 8-Bromo-cAMP and the PKA inhibitor H-89 dihydrochloride on nociceptive thresholds to mechanical stimulation in six adult sheep to define further the role of cAMP in spinal nociception. Treatment with 420 nmol 8-Br-cAMP induced significant hypoalgesia to noxious stimulation, while a 10-fold higher dose (4.2 micromol) induced mechanical hyperalgesia. Both of these behaviours were blocked by H-89 (38-380 nmol). Treatment with high dose H-89 (380 nmol) alone significantly increased nociceptive thresholds. These results demonstrate that activation of the cAMP-PKA signalling pathway modulates acute nociceptive events in spinal cord in a biphasic manner, and suggest that significant tonic activity exists in this pathway.

Measurement of cyclooxygenase inhibition in vivo: a study of two non-steroidal anti-inflammatory drugs in sheep.

The anti-inflammatory effects of the non-steroidal anti-inflammatory drugs phenylbutazone (PBZ) and flunixin meglumine (FM) and the relationship between the effects and drug concentration in vivo were studied using a subcutaneous tissue-cage model in sheep. Intracaveal injection of carrageenan induced prostaglandin (PG) E2 production in tissue-cage exudate (maximal concentration, 101 nM) with significant increases in white blood cell (WBC) numbers, skin temperature over the inflamed cage and exudate leukotriene B4 (LTB4) concentration (P < 0.05). Intravenous PBZ, 4.4 mg kg-1 produced mild inhibition of exudate PGE2 generation (10%), but greater inhibition of serum TXB2 (75.3%). The IC50 for TXB2 was 36.0 microM. Phenylbutazone did not alter effects on skin temperature, WBC numbers or exudate LTB4 concentrations. Intravenous FM, 1.1 mg kg-1, significantly inhibited carrageenan-induced exudate PGE2 formation (Emax, 100%, IC50, < 0.4 nM) and serum TXB2 generation (Emax, 100%, IC50, 17 nM) for up to 32 h. Flunixin meglumine significantly inhibited the rise in skin temperature but had a limited effect on exudate WBC. Phenylbutazone and FM have distinct effects on carrageenan-induced cyclooxygenase (COX-2) and platelet COX (COX-1). Flunixin meglumine was a more potent COX inhibitor than PBZ and was more selective for the inducible form of COX in vivo.

Effect of flunixin meglumine on the thresholds to mechanical stimulation in healthy and lame sheep.

The antinociceptive effect of flunixin meglumine was assessed in healthy and lame sheep by using a noxious mechanical stimulus. Sheep suffering from the chronically painful condition, footrot, have previously been shown to have lower thresholds to noxious mechanical stimuli than healthy animals. In the present study, 22 sheep suffering from footrot did not have a lower mean mechanical threshold than 25 matched healthy animals, but it was significantly greater than that recorded from eight experimental sheep (5.0 [2.5], 4.9 [2.1] and 3.0 [1.0] Newtons, respectively). Doses of 1.0 or 2.0 mg kg-1 of flunixin meglumine had no effect on the thresholds to noxious mechanical stimulation in either experimental sheep tested over six hours, or in lame sheep tested over a period of 30 minutes. The repeated administration of flunixin to sheep suffering from footrot over a period of three days reduced their thresholds to noxious mechanical stimulation.

Pharmacokinetics of propofol as an induction agent in geriatric dogs.

The pharmacokinetics of propofol were investigated in six unpremedicated dogs aged between eight-and-a-half and 10.5 years. After the induction of anaesthesia with a bolus dose of 5 mg kg-1 propofol administered intravenously, the trachea was intubated and anaesthesia was maintained with halothane in a gas mixture of oxygen and nitrous oxide. The mean (sd) t1/2 beta was 83.9 (14.8) minutes, the mean (SD) residence time was 99.0 (10.8) minutes, the mean body clearance was 34.5 (12.1) ml kg-1 minute-1 and the mean volume of distribution at steady state was 3864 (1647) ml kg-1. The dose of propofol was lower than that recommended in the data sheet (6.5 mg kg-1) and was associated in some of these dogs over eight years of age with post-induction apnoea. The clearance of propofol was also slower than that previously reported in young dogs. These results have implications for infusion rates when propofol is used to maintain anaesthesia in dogs.

Effects of non-steroidal anti-inflammatory drugs on the hyperalgesia to noxious mechanical stimulation induced by the application of a tourniquet to a forelimb of sheep.

A tourniquet was used in conjunction with a mechanical threshold testing device to investigate the suitability of the technique for the investigation of analgesic drugs in sheep. The changes to the mechanical thresholds to noxious stimulation during and after the inflation of a pneumatic tourniquet on a limb were recorded, and the influence of pre-treatment with two non-steroidal anti-inflammatory drugs was studied. Fentanyl, an opioid agonist with known analgesic properties in sheep, was used as a positive control. The tourniquet significantly reduced the mechanical thresholds on the ipsi- but not the contralateral limb. Pretreatment with either flunixin meglumine or carprofen attenuated the development of mechanical hyperalgesia, and fentanyl initially caused a significant anti-nociceptive effect. The time to aversion was not significantly different between the treatments. These results suggest that hyperalgesia induced by a tourniquet may be a useful technique for the investigation of the anti-nociceptive effects of analgesic drugs in sheep.

Pharmacokinetics of carprofen administered intravenously to sheep.

Carprofen was administered intravenously to sheep at two dose rates (0.7 and 4.0 mg kg-1), and the pharmacokinetics of the drug studied. Plasma concentrations of the drug were measured by high performance liquid chromatography. Carprofen had a small volume of distribution (Vd[area], 95.5 and 118.4 ml kg-1), a prolonged elimination half-life (t1/2 beta, 26.1 and 33.7 hours) and a slow body clearance rate (Clb, 2.5 ml kg-1 h-1) in sheep.

Pharmacokinetics of propofol infusions, either alone or with ketamine, in sheep premedicated with acepromazine and papaveretum.

The pharmacokinetics of propofol were investigated in two groups of five Scottish blackface sheep undergoing surgery for the implantation of subcutaneous tissue pouches. After premedication with acepromazine and papaveretum, anaesthesia was induced with either propofol at 4 mg kg-1 intravenously (group 1) or with a mixture of propofol at 3 mg kg-1 and ketamine at 1 mg kg-1 intravenously (group 2). Anaesthesia was maintained with a variable infusion rate of either propofol alone (group 1) or propofol and ketamine (group 2). Both regimens produced satisfactory conditions for superficial surgery of the body surface. The mean (SD) duration of anaesthesia was 64.8 (3.1) minutes for group 1 and 60 (0) minutes for group 2; the mean total dose of propofol given to the sheep in group 1 was 801 (84) mg, and the sheep in group 2 received 470 (46) mg of propofol and 267 (30) mg of ketamine. The mean elimination half-life of propofol was 56.6 (13.1) minutes in group 1 and 50.3 (21.4) minutes in group 2; the mean volume of distribution at steady state was 1.037 (0.480) litre kg-1 in group 1 and 1.515 (0.939) litre kg-1 in group 2; the mean body clearance was 85.4 (28.0) ml kg-1 min-1 in group 1 and 128.0 (35.0) ml kg-1 min-1 in group 2; the mean residence time corrected for a bolus injection was 12.1 (4.2) minutes in group 1 and 11.9 (6.6) minutes in group 2; for the infusion, the mean residence time was 72.1 (4.2) minutes in group 1 and 69.9 (7.9) minutes in group 2. There were wide variations in the blood propofol concentrations reached in individual sheep by using this standard dosing regimen. All the sheep recovered quickly from anaesthesia; the mean times to extubation, sternal recumbency and standing for the animals in group 1 were 2.8 (0.4), 6.3 (1.2) and 10.9 (1.6) minutes from the end of the infusion, and the times for group 2 were 5.3 (0.9), 11.2 (1.7) and 15.1 (2.2) minutes.

Plasma disposition, faecal excretion and in vitro metabolism of oxibendazole following oral administration in horses.

Oxibendazole (OBZ) was administered to eight horses at an oral dose of 10 mg kg(-1) bodyweight each. Parent OBZ could only be detected in plasma at the 0.5 and 1.0 hours post administration sampling times and the mean maximum plasma concentration was 0.008 microg ml(-1). Parent OBZ was detected in faeces between 12 and 72 hours after administration and the highest dry faecal concentration was detected at 24 hours. An unidentified metabolite was detected in plasma between 0.5 and 72 hours. The unidentified metabolite in the plasma of treated horses corresponded to the second eluted metabolite in the in vitro study. Metabolism of OBZ to its metabolite in vitro was significantly inhibited by co-incubation with the cytochrome P450 inhibitor piperonyl butoxide. These results indicated that first-pass metabolism decreases OBZ bioavailability in horses. The in vitro metabolism of OBZ was significantly inhibited by piperonyl butoxide and this could be utilised to extend the exposure of nematodes to the parent molecule.

Methods for the isolation, culture and characterisation of equine pulmonary artery endothelial cells.

Equine endothelial cells were isolated from the pulmonary artery by enzymatic digestion and grown to confluency. The cells were characterised by positive immunofluorescent staining for von Willebrand factor and NADPH-diaphorase staining for nitric oxide synthase. Measurements of endothelins indicated that there were significant release rates from the cells for up to six hours. Measurements of intracellular calcium concentration showed that the application of bradykinin caused a transient increase in calcium concentration with similar characteristics to those observed in other endothelial cell preparations. These tests verify the endothelial character of these cells and establish the method as a reliable means of producing a primary culture of equine endothelial cells.

Effects of aspirin on xylazine-induced hypoxaemia in sheep.

Aspirin (10 mg kg-1) administered intravenously to conscious sheep four hours before intravenous xylazine injection (50 micrograms kg-1), failed to abolish or attenuate the hypoxaemic effect of xylazine in this species. Serum thromboxane levels measured in one animal revealed that aspirin administered in this way reduced serum thromboxane levels by 95 per cent. Xylazine (3 x 10(-5) M--4 x 10(-3) M) failed to induce platelet aggregation in vitro. It appears that the mechanism whereby xylazine causes arterial hypoxaemia in sheep does not involve a cyclo-oxygenase-dependent aggregation of platelets.

Changes in the Doppler waveform of the ovine femoral artery following infusion of vasoactive agents: a preliminary study.

The aim of this study was to investigate whether femoral artery pulsatility index (PI) can be used as an indicator of vasomotor tone in the digit of an ungulate species by measuring the change in PI induced by infusion of vasoactive agents in halothane-anaesthetised sheep. Intra-arterial infusion of the vasoconstrictor phenylephrine (9 microg min(-1)) tended to increase waveform PI (3.89-6.24, n=4, P=0.100). Infusion of a low dose (3 microg min(-1)) of the vasodilator sodium nitroprusside did not alter femoral artery PI, however infusion of a higher dose (30 microg min(-1)) tended to increase PI (5.39-6.70, n=5, P=0.059). During these studies heart rate, mean ABP and p(a)CO(2) did not change significantly. The tendency for PI to increase in response to vasodilation was unexpected and the mechanism involved is unclear. It appears that femoral artery PI cannot be used to predict vasomotor tone in the digit of the anaesthetised sheep.