Sl Smith

Accumulation and depletion of liver copper stores in dairy cows challenged with a Cu-deficient diet and oral and injectable forms of Cu supplementation.

Abstract AIM: To quantify the capacity of the liver to respond to a Cudeficient diet and various forms of Cu supplementation in dairy cows with high Cu status. METHODS: Sixty non-pregnant, non lactating mixed-age Freisian dairy cows were fed baled silage for 116 days, containing 5.8 mg Cu/kg dry matter (DM), that provided an inadequate Cu intake. They were either unsupplemented (Control); or treated thrice weekly orally with solutions containing the equivalent of 150 mg Cu/day as copper sulphate pentahydrate (CS), copper amino chelate (CAC), or copper glycinate (CG); 16 g Cu as Cu0 wire particles, administered in an intra-ruminal bolus on Day 0; or 100 mg Cu as calcium copper edetate, administered by S/C injection on Days 0 and 58. On Days -5, 14, 28, 58, 86 and 116, liver biopsies and blood samples were obtained for determination of Cu. RESULTS: Mean initial concentrations of Cu in liver for all groups was 827 (SE 42) µmol/kg fresh tissue. In control cows, this decreased to 552 µmol/kg on Day 116, and averaged 670 µmol/kg over the length of the trial. Oral forms of Cu supplementation increased overall mean concentrations in liver to 960 (SE 79), 1,050 (SE 81) and 1,100 (SE 84) µmol/kg for CS, CAC and CG, respectively, but there was no difference between form of supplement. Mean concentrations were significantly increased by bolus treatment, but not by injection. Concentrations of Cu in serum in all groups decreased from 12.1 (SE 0.3) to 10.4 (SE 0.6) µmol/L by Day 116, with no differences due to treatments. The initial concentration of Cu in liver significantly affected the rate of accumulation of Cu among cows supplemented orally. In cows with an initial concentration <1,100 µmol/kg, the average increase was 4.1 µmol/kg fresh tissue/day, whereas rates were variable, even negative, when initial concentrations were >1,100 µmol/kg. Release of Cu over 116 days in cows given the bolus was calculated to be equivalent to an oral intake of CS of 106 mg Cu/day. CONCLUSIONS: In dairy cows with a high Cu status fed a Cu-deficient diet, responses to supplementation with two chelated forms of Cu administrated orally were similar to those due to CS. Copper as CuO was about 77% as effective as oral supplementation with chemically similar inorganic Cu. Initial concentrations of Cu in liver influenced the rate of accumulation following oral supplementation. CLINICAL RELEVANCE: Changes in concentrations of Cu in liver were readily observed following oral supplementation over a wide range of initial concentrations, whereas these changes could not be detected in serum. The liver is thus a good index of the Cu status of dairy cows.

Copper deficiency in sheep: An assessment of the relationship between concentrations of copper in serum and plasma

Abstract AIM: To assess the relationship between concentrations of copper in serum and plasma in sheep. METHODS: Concentrations of Cu were measured in paired serum and heparinised plasma samples collected from 110 sheep in nine flocks. Linear regression was used to evaluate whether flock or gender had a significant effect on the association between concentrations of Cu in serum and plasma. The individual results for concentrations of Cu in serum were then compared with those from plasma, using correlation and limits of agreement plotting. RESULTS: Concentrations of Cu in serum ranged from 7.3 to 22 (mean 14.0) µmol/L, while concentrations in plasma ranged from 9 to 27 (mean 16.3) µmol/L. On average, concentrations of Cu in serum were 2.3 µmol/L lower than in plasma. Over the range of values seen in this study, concentrations of Cu in plasma and serum were significantly correlated (r=0.89), and mean concentrations in serum were 87% of those in plasma. There was no effect of flock or gender on the relationship between concentrations of Cu in serum and plasma. Despite the significant correlation, there was marked variability between individual samples in the proportion of Cu that was lost during clotting, with the 95% limits of agreement for serum Cu ranging from 70 to 104% of the plasma concentration. CONCLUSIONS: As in cattle, the individual variability in the loss of Cu during clotting in sheep is too great for concentration of Cu in serum to be used as a substitute for that in plasma. CLINICAL RELEVANCE: When assessing the blood Cu pool as part of the diagnosis of Cu-responsive disease in sheep, the concentration of Cu in plasma should be measured in preference to that of serum. We suggest that a range of 4.5 to 9 µmol/L in plasma be used to define marginal Cu status in sheep.

Ram and buck management

Careful management is necessary to ensure the reproductive success in any small ruminant farm and to maximise the productive longevity of rams and bucks. Rams and bucks are frequently overlooked outside of the breeding period, but year-round attention to nutrition, parasite control and general disease control is important in keeping them healthy and sound for breeding. Pre-mating soundness examinations are an inexpensive and relatively easy way to assess the potential ability of a ram or buck to perform during the breeding period and should be incorporated annually into the management of any flock. During the breeding period, careful thought should be given to the appropriate use of males, and the effectiveness of mating should be monitored.

Detection of Neospora caninum DNA in semen of experimental infected rams with no evidence of horizontal transmission in ewes.

Recent reports from New Zealand indicate Neospora caninum has a possible role in causing abortions in sheep. Transmission of N. caninum via semen has been documented in cattle. This study aimed to investigate if horizontal transmission through semen was also possible in sheep. Initially, 6-month old crossbred ram lambs (n=32), seronegative to N. caninum, were divided into 4 equal groups. Group 1 remained uninoculated whilst the remainder were inoculated with N. caninum tachyzoites intravenously as follows: Group 2 - 50 tachyzoites; Group 3 - 10(3) tachyzoites; Group 4 - 10(7) tachyzoites. Semen samples were collected weekly for 8 weeks for the detection of N. caninum DNA and quantified using quantitative PCR (qPCR). Plasma collected 1 month post-inoculation was subjected to ELISA (IDEXX Chekit) and Western blot. At 2 weeks post-infection, three rams from Group 1 (uninoculated) and three rams from Group 4 (10(7)tachyzoites/ml) were mated with two groups of 16 ewes over two oestrus cycles. Ewe sera collected 1 and 2 months post-mating were tested for seroconversion by ELISA and Western blot. All experimentally infected rams seroconverted by 1 month with ELISA S/P% values ranging from 11% to 36.5% in Group 2, 12-39.5% in Group 3 and 40-81% in Group 4. However, none of the ewes mated with the experimentally infected rams seroconverted. For the Western blot, responses towards immunodominant antigens (IDAs) were observed in ram sera directed against proteins at 10, 17, 21, 25-29, 30, 31, 33 and 37 kDa. Rams in Group 2, 3 and 4 were noted to have at least 3 IDAs present. None of the ewes showed any of the 8 prominent IDAs except for the one at 21 kDa which was seen in 30 out of 32 ewes in both groups. N. caninum DNA was detected intermittently in the rams semen up to 5 weeks post-inoculation with the concentrations ranging from that equivalent to 1-889 tachyzoites per ml of semen. Low concentrations of N. caninum DNA were also detected in the brain tissue of two rams (Groups 1 and 4). These results suggest that although N. caninum DNA can be found in the semen of experimentally infected rams, the transmission of N. caninum via natural mating is an unlikely event.

Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand.

Abstract AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johnes disease confirmed histologically. METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johnes disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johnes disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes. RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johnes disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johnes disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johnes disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3–142.0; p<0.001). CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johnes disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood. CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johnes disease.

The effect of the maturity and prior breeding activity of rams and body condition score of ewe hoggets on the reproductive performance of ewe hoggets

Abstract AIM: To determine the effect of age and prior use of mature rams at a given ram-to-ewe ratio, and the effect of body condition on breeding performance and pregnancy rate of ewe hoggets. METHODS: Ewe hoggets (n=733) aged 7–8 months were weighed and their body condition scored, then randomly assigned to one of three treatment groups (Day 0) and joined with either four two-tooth rams (20 months of age) not used previously (n=244; Two-tooth), four mixed-aged mature rams that had not been used earlier in the season (n=244; Mature-fresh), or four mixed-aged mature rams that had been used with mature ewes immediately prior to joining with hoggets (n=245; Mature-used). The breeding period was 34 days. Ewe hoggets were identified as having been marked during the first 17 days only, during both 17-day periods, during the second 17 days only, or not marked. Hoggets were re-weighed on Day 34, and pregnancy status determined using ultrasound on Day 92. The breeding soundness of the rams was assessed on Days −34 and −1. RESULTS: Semen samples obtained from the rams did not differ significantly in any of the parameters measured (p>0.05). Ewe hoggets joined with Mature-fresh rams were less likely (p<0.05) to be marked in the second 17 days of breeding only than those joined with either Two-tooth or Mature-used rams. No other breeding parameters were affected by breeding group (p>0.05). Hoggets marked in the first 17 days only were heavier (p<0.05) at Day 0 than those marked in the second 17 days only or not marked. Hoggets diagnosed as twin-bearing were heavier (p<0.05) than non-pregnant or single-bearing hoggets. Those hoggets marked in the first 17 days only had a greater (p<0.05) body condition score (BCS) than those marked in the second 17 days only or not marked at all. These differences were no longer apparent after correction for liveweight (LW).Correction for LW at Day 0 or change in LW during the breeding period did not affect the results for breeding performance. CONCLUSIONS: Under the conditions of this study, two-tooth rams and mature rams that had been used previously were just as suitable as mature rams that had not been used previously for breeding with ewe hoggets. Further studies are warranted to verify this result. The re-use of rams without reducing breeding performance would reduce breeding costs and may make breeding hoggets a more viable option for farmers. The BCS of ewe hoggets affected breeding performance, and can thus be used to identify those animals most suitable for breeding.

The role of liver Cu kinetics in the depletion of reserves of Cu in dairy cows fed a Cu-deficient diet.

Abstract AIM: To determine how the concentration of Cu in liver affects the rate of depletion of that Cu when cows are fed a Cu-deficient diet under experimental conditions, and to mathematically model the rate of depletion of Cu over time. METHODS: In June 2010, 25 non-lactating Friesian cows were assigned to three groups such that initial mean concentrations of Cu in liver were 265, 534 and 1,486 μmol Cu/kg fresh tissue (Day 0). All cows were managed as a single group and fed a Cu-deficient diet of primarily baled silage. No mineral Cu supplements were given. Liver biopsies were collected from cows on Days 0, 53, 98 and 161 to determine concentrations of Cu. At about the same time, samples of silage and pasture herbage were collected to determine Cu, Mo and S concentrations. RESULTS: Median concentration of Cu in silage was 6.5 (min 6, max 9) mg/kg DM. Concentration of Cu in liver decreased in all groups (p<0.001), over the duration of the study. The amount of Cu depleted from liver was greater in groups that started the study with higher initial concentrations of Cu in liver. The rate of decline followed exponential first-order kinetics with an elimination rate constant k of 0.0057 (CI 95%=0.0039–0.0074), meaning that about 0.57% of liver Cu reserves were depleted each day. For individual cows this loss amounted to 0.1–14 μmol Cu/kg liver/day. CONCLUSIONS AND CLINICAL RELEVANCE: Depletion of Cu from liver was dependent on initial concentration of Cu. These results can be used to predict how long an unsupplemented herd will remain in adequate Cu status, which adds confidence to decisions about when Cu supplementation should be withdrawn or reinstated. Cows with high concentrations of Cu in liver can maintain adequate Cu status for months without supplements. Intake of less Cu and more Mo would increase the rate of depletion, and seasonal factors would also have some influence.

The impact of high zinc intake on the copper status of dairy cows in New Zealand

Abstract AIM: To determine the effect of a high Zn intake on the concentration of Cu in the liver of dairy cows. METHODS: Sixty non-pregnant, non-lactating, mixed-age Friesian dairy cows were randomised into four groups: unsupplemented control; thrice weekly oral treatment with 350 mg Cu (equivalent to 150 mg Cu/day) as CuSO4.5H2O; a ZnO bolus releasing 6.628 g Zn/day given on Days 0 and 28; and oral treatment with Cu and ZnO boluses. The cows were fed baled silage for the duration of the study (56 days). On Days -12, 14, 28, 42 and 56 liver biopsies were collected for determination of concentrations of Cu, and blood samples for measurement of Cu and Zn in serum. RESULTS: Mean concentrations of Cu in the liver of unsupplemented control cows decreased from an initial 717 (SE 108.5) to 396 (SE 88.0) µmol/kg fresh tissue on Day 28, and then increased to 542 (SE 105.3) µmol/kg fresh tissue on Day 42, before decreasing to 434 (SE 81.6) µmol/kg fresh tissue on Day 56. Administration of ZnO resulted in no change in the concentration of Cu in liver compared with controls. Supplementing with 150 mg Cu/day significantly increased the concentration of Cu in liver, compared with controls, to 1,074 (SE 105.2) µmol/kg fresh tissue on Day 56. Treatment with ZnO significantly reduced concentrations of Cu in liver on Day 56, compared with Cu-supplemented cows, to 786 (SE 107.7) µmol/kg fresh tissue. The treatments had no effect on concentrations of Cu in serum. In cows given the ZnO, mean concentrations of Zn in serum increased from 17 (SE 0.6) on Day 0 to 30 (SE 1.7) µmol/L on Day 14, then decreased to 20 (SE 1.3) µmol/L on Day 28, increased to 40 (SE 2.9) µmol/L on Day 42, then decreased to 24 (1.16) µmol/L on Day 56. In cows given ZnO plus Cu, concentrations of Zn in serum were significantly greater than in cows only given ZnO on Day 41 (46 (SE 2.71) vs 35 (SE 3.23) µmol/L, respectively). Mean concentrations of Cu, Mo and Zn in the baled silage were 4.4, 0.5 and 21 mg/kg dry matter (DM), respectively. CONCLUSIONS: High Zn intakes used in the prevention of facial eczema in dairy cows had little effect on concentrations of Cu in liver when the Cu intake was low, but decreased the efficacy of a Cu supplement by about 50%. CLINICAL RELEVANCE: When using Zn as a prophylactic treatment for facial eczema the level of Cu supplementation should be adjusted, which may mean that in some situations it is recommended not to supplement cows with Cu.

Seroconversion and semen shedding in rams experimentally infected with Brucella ovis

AIM: To determine the time taken for rams to develop antibodies to Brucella ovis in serum, shed B. ovis in semen and develop lesions of epididymitis following infection with B. ovis. METHODS: Fifteen 19-month-old rams were artificially infected with B. ovis by inoculation of infected semen onto the nasal and rectal mucus membranes (Day 0). Serum was collected from each ram at 2 to 8-day intervals and tested at commercial laboratories using a complement fixation test (CFT) and an ELISA. Cut-off values for the CFT were 0–4/4 negative; 1/8–3/8 suspicious and 4/8–4/128 positive, and for the ELISA were <10% negative; ≥10 to <50% suspicious and ≥50% positive. Selected serum samples were also tested using a gel diffusion test (GDT). At 7 to 8-day intervals semen was collected for bacterial culture and the scrotal contents were palpated to identify lesions of epididymitis. The study was terminated after 56 days. RESULTS: On Day 28 B. ovis was isolated from the semen of one ram and by Day 49 it was isolated from the semen of 10 rams. All 10 rams had suspicious or positive ELISA or CFT titres by Day 36 and 56, respectively. The GDT results were all negative on Day 36 and in general did not become positive in individual rams until 7–28 days after semen shedding commenced. Epididymitis was detected in one ram on Day 36; by Day 56 eight rams had epididymitis detectable by scrotal palpation. CONCLUSIONS: The B. ovis ELISA test identified infected rams at an earlier stage than the CFT; this was at 19–36 days after exposure. Rams can begin shedding B. ovis in semen as early as 28 days after exposure and lesions of epididymitis develop as early as 36 days after exposure. CLINICAL RELEVANCE: During a test and slaughter campaign for the control of B. ovis, the most appropriate serological re-testing interval is likely to be around 28 days (4 weeks) using the ELISA with or without the CFT, although caution is required in interpretation of “suspicious” ELISA results. Following a B. ovis breakdown, two negative CFT or ELISA tests 60 days apart are recommended to confirm freedom from infection, supporting current guidelines.

Adaptation of a commercial ELISA to determine the IgG avidity in sheep experimentally and naturally infected with Neospora caninum

Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.