Salivary cortisol as an indicator of stress in sheep (Ovis ovis)

Recent work^p(1)(2) has suggested that measuring salivary cortisol may provide a noninvasive measurement of stress-induced changes. A noninvasive collection technique is particularly useful in stress and welfare studies because the technique may not impose additional stress, as would a more invasive technique such as venepuncture.

We have developed a method that allows saliva collection from sheep for subsequent cortisol analysis and shows unambiguous stress related changes. Saliva was collected by use of an absorbent strip of cloth (Chux, Nationalpak Ltd, Auckland) attached to the end of a rod. A swab was cut from the cloth to the dimensions 260 x 40 mm. The rod dimensions were 1 mm diameter, 450 mm long with a 10 mm loop at one end for swab attachment. The sheep were manually restrained while the swab was placed in the mouth and the animal allowed to chew on it until the swab was saturated with saliva (generally 10–40 seconds). Saliva was expressed from the swab using a syringe (one per animal). If the saliva sample volume was less than 1 ml, the collection procedure was repeated using the same a to yield a greater sample volume. Saliva samples were stored at -20 °C within 1 hour of collection.     

Basal and stress response cortisol levels and stress avoidance learning in sheep (Ovis ovis)

Basal and stress related corticosteroid levels show complex patterns of relationship to age, social status and aggressive behaviour ^{(1)(2)(3)(4)(6)}. I have investigated basal and stress related cortisol levels in sheep subjected to a model of learned stress avoidance, the results of which suggest that factors of stress avoidance learning ability and experience also contribute to cortisol levels.     

Salivary and plasma cortisol as an index of stress in goats

Total assayable cortisol in plasma was highly correlated (r = 0.97) with physiologically active free cortisol in plasma after routine management procedures in 1- to 3-weeks-old goats. Transport of adult goats caused significant increases (P less than 0.001) in free cortisol in saliva and in free and total cortisol in plasma. No difference (P greater than 0.05) between concentrations of free cortisol in saliva and in plasma was apparent before or after transport. The results demonstrated that the salivary cortisol method is a useful measure of stress in adult goats, and that the relationship between free and total cortisol in plasma, and the adrenocortical response to transport, appear to be similar in sheep and goats.     

Peripheral concentrations of cortisol as an indicator of stress in the pig

Twenty-four crossbred gilts (7 to 9 months old) were exposed to 3 different stressors to evaluate changes in peripheral concentrations of the adrenal hormone cortisol. Gilts were confined in a box for 1 hour, electrically stimulated for 6 minutes, or heat-stressed for 6 hours; these stressors were imposed for 3 consecutive days. Blood samples were collected repeatedly, and serum cortisol concentrations were determined by radioimmunoassay. Peak cortisol concentrations for pigs in the box, electrically stimulated, and heat-stressed were 67.8 +/- 9.9 ng/ml (mean +/- SEM), 43.2 +/- 5.8 ng/ml, and 25.2 +/- 3.0 ng/ml greater than the control concentrations. The times at which these peaks occurred varied with each stressor. Overall serum cortisol concentrations decreased from day 1 to day 3, but the response to a given stressor was not diminished. Our data substantiate the use of cortisol as an indicator of stress in swine and emphasize consideration for the interval between exposures to stressors.     

Salivary cortisol as a stress parameter in piglets]

The relationship between salivary and plasma levels of total and free cortisol was monitored in 97 male piglets, aged two to four weeks, subjected to castration. Samples were taken 10 minutes before (basal value) as well as one, two, three, four and 24 hours post castration and at the same time intervals from a control group of 17 animals which did not undergo surgery. Simultaneously to blood (indwelling catheter) withdrawing saliva was collected by two cotton swabs. Cortisol levels were measured by radioimmunoassay (RIA). A highly significant increase in total, free and salivary cortisol was found within the first four hours after castration compared to the control group. The percentage increase one hour after castration above basal values was highest in free plasma cortisol (21.08 +/- 2.03 nmol/l vs. 61.26 +/- 4.16 nmol/l; 290.6%), and lowest in total plasma cortisol (177.33 +/- 9.69 nmol/l vs. 374.09 +/- 18.21 nmol/l; 211.0%), whereas salivary cortisol showed an 255.7% increase (10.46 +/- 1.03 nmol/l vs. 26.75 +/- 1.93 nmol/l). Total cortisol included 11.9-16.4% free cortisol. Salivary cortisol concentration was between 5.9% and 7.5% of the total plasma cortisol concentration. The highest correlation between total plasma cortisol and salivary cortisol occurred one hour after castration (r = 0.57; p < 0.01). The correlation between free and salivary cortisol was lowest for basal values (r = 0.27; p < 0.05), whereas correlations for the remaining time points were highly significant (0.41 < or = r < or = 0.61; p < 0.01). For the control group significant correlations were found between salivary and total plasma cortisol (0.58 < or = r < or = 0.89; p < 0.05) and between free and salivary cortisol (0.63 < or = r < or = 0.92; p < 0.05). The present work indicates that the measurement of salivary levels of cortisol reflects the concentration of this hormone in plasma samples of piglets.     

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β₂ integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils.     

Differential pulmonary immunopathology of domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) with Mycoplasma ovipneumoniae infection: A retrospective study

Mycoplasma ovipneumoniae is a respiratory pathogen that impacts domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). BHS are reported to be more susceptible than DS to developing polymicrobial pneumonia associated with M. ovipneumoniae infection. Using formalin-fixed paraffin-embedded tissues, we performed a retrospective study investigating the pulmonary immune response of DS and BHS to M. ovipneumoniae infection. M. ovipneumoniae infected DS exhibited a more robust and well-organized BALT formation as compared to BHS. Digital analysis of immunohistochemical chromogen deposition in lung tissue was used to quantitate T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. A significant interaction of species and infection status was identified for CD3, CD163, and IL-17. BHS had a greater increase in bronchiolar CD3 and bronchiolar and alveolar CD163 with infection, as compared to DS. BHS had an increase in bronchiolar associated lymph tissue (BALT) and alveolar IL-17 with infection, while these remained similar in DS regardless of infection status. IL-17 in respiratory epithelium of bronchi and bronchioles comparatively decreased in DS and increased in BHS with infection. These data begin to define the interspecies differential immune response to pulmonary M. ovipneumoniae infection in DS and BHS and provide the first investigations of respiratory epithelium-associated IL-17 in ovine.     

Approaches to vaccines for Psoroptes ovis (sheep scab)

A brief outline of the history of sheep scab in the UK is presented together with the current chemical methods used for its control and problems associated with these. Possible alternative approaches to control are discussed, as are selected aspects of the physiology of Psoroptes ovis and the pathogenesis of the scab lesion from the perspective of control through immunisation. Evidence is provided that immunity to the disease can indeed be acquired, both naturally after a previous infection and following inoculation of mite proteins in adjuvant. These results support the view that control by vaccination may be possible, although little is known to date about the antigens involved or the mechanism of protection.     

Evaluation of strategies for control of sheep lice (Damalinia ovis) with an epidemiological model

A model was constructed to integrate the epidemiology and economics of control of sheep lice in Victoria and provide an objective basis for comparing lice control strategies. The strategies evaluated included different allocations of Department of Agriculture and Rural Affairs (DARA) resources for detection and supervision of infected flocks and for extension to enhance owner detection rates, change treatment practices or promote the adoption of disease prevention measures. Changes in prevalence and the net present cost of sheep lice to the industry were calculated over a 10-year period. The model predicted that the most profitable lice control strategy was to discourage routine annual treatment, promote treatment on detection, and encourage the adoption of lice prevention measures. The number of infested flocks undertaking eradication programs under DARA supervision had little effect on the prevalence or net cost of lice to Victoria.     

Maternal behaviour in sheep (Ovis aries) following administration of opioid agonists

AIMS: As kappa(kappa)-opioids have marked effects on pain threshold in sheep during oestrus, late pregnancy and after birth, a study was undertaken to determine if kappa -opioids also had other roles during these states. METHODS: Opioid agonists (GR89696, DAMAGO) were administered into either the lateral hypothalamus (LH) or amygdala of non-pregnant sheep (n = 5) or pregnant sheep (n = 15) in late pregnancy (n = 5) within 12 h of birth (n = 5) or in the third week after birth (n = 5). Cortisol, behavioural and electrocardiographic responses to an auditory stimulus were assessed with and without drug administration, as was feed consumption and suckling events. RESULTS: In non-pregnant sheep, DAMAGO injected into either the LH or amygdala had little effect on the response to the auditory stimulus but when injected into the LH did decrease food consumption. GR 89696 had no obvious effects. In late pregnancy, cortisol and heart rate responses to the auditory stimulus were significantly reduced compared to the non-pregnant animals irrespective of treatment. This was also the case in the third week after birth. Immediately post-partum cortisol responses were closer to that seen in the non-pregnant animal. In late pregnancy and both post-partum periods, GR 89696 in the LH significantly increased feed consumption while DAMAGO had no significant effects. GR 89696 injected into the amygdala reduced the behavioural response to the auditory stimulus in late pregnancy and both post-partum periods. CONCLUSION: Changes in sensitivity to opioids occur in both the amygdala and LH with pregnancy and after birth. These may influence maternal behaviours and feed intake.     

Test of the sheep ked Melophagus ovinus (L) as a vector of Anaplasma ovis Lestoquard

The sheep ked Melophagus ovinus was evaluated as a possible vector of Anaplasma ovis. In 4 tests, 45 to 293 keds were transferred from sheep with acute anaplasmosis, low level parasitemia, or carrier state of anaplasmosis to individual splenectomized ewes. Keds were confined in stockinette cages attached to the dorsal midline of the recipient hosts for 10 days. Throughout the 90-day observation periods after the keds were transferred, no clinical, serologic, or hematologic evidence of anaplasmosis was detected in any of the ked-recipient ewes. Results indicate that sheep keds probably are not mechanical or biological vectors of the ovine anaplasmosis organism.     

Ceruloplasmin as an indicator of copper status in cattle and sheep.

The relationship between ceruloplasmin, a metalloenzyme with oxidase activity, and copper was investigated in cattle and sheep. The oxidase activity of ceruloplasmin correlated closely with the serum or plasma copper concentrations in cattle. The respective correlation coefficients were 0.83 and 0.60. In sheep serum, a correlation coefficient of 0.92 was obtained. In each instance, the relationship remained linear from the deficient to the high normal ranges of copper. Comparison of the linear regression relationships indicated the ceruloplasmin activity in bovine serum was statistically lower than the activity in bovine plasma (P less than 0.0001), through the intercepts from the regression lines of the two relationships were similar (P = 0.412). Comparisons of ovine and bovine serum-ceruloplasmin relationships indicated that a significant species difference was present. Ovine ceruloplasmin activity increased more rapidly as compared to the corresponding bovine activity over the range of copper concentrations investigated (P less than 0.0001). The intercept from the ovine regression relationship was also lower (P less than 0.0001). A correlation coefficient of 0.35 was observed between the serum ceruloplasmin activity and hepatic copper concentrations in cattle indicating that the mathematical relationship was not as well defined. Ceruloplasmin activity appears to correlate more closely with serum or plasma copper concentrations as compared to corresponding liver copper concentrations.