Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Pharmacological characterisation of the electrically evoked release of monoamines from chicken brain in vitro

1. A study was conducted to develop an in vitro model for examining the basal and electrical-stimulation-induced release of [3H]monoamines from chicken hyperstriatal neurones in order to demonstrate the presence of presynaptic autoreceptors for the three main monoamine transmitters: noradrenaline, dopamine and 5-HT. 2. Two sets of experiments were carried out: the first was to evaluate the effect of calcium and tetrodotoxin (TTX, sodium channel conductance inhibitor) in order to demonstrate that evoked release of monoamines was a consequence of exocytotic processes; the second to investigate the effect of selective agonists and antagonists on neurotransmitter release. 3. Ross and Cobb broiler chickens of either sex (approximately 7 to 8 weeks old) were used. Slices of hyperstriatal tissue were preincubated with [3H]noradrenaline, [3H]dopamine or [3H]5-hydroxytryptamine (5-HT), washed, perfused and electrically stimulated at three time points (S1, S2 and S3) which released [3H]noradrenaline, [3H]dopamine and [3H]5-HT, as determined by scintillation spectrometry. 4. When calcium was removed from, or TTX added to, the superfusion medium prior to and including the second period of electrical stimulation (S2) the evoked releases of [3H]noradrenaline, [3H]dopamine and [3H]5-HT at S2 were abolished. 5. In the presence of the selective alpha2-adrenoceptor agonist UK 14304 during the S2 period, the S2/S1 ratio was lower than the control ratio due to a reduction in the stimulated release of [3H]noradrenaline. The selective alpha2-adrenoceptor antagonist RX 821002 blocked the UK 14304-induced reduction of evoked release and the S2/S1 ratio was similar to the control ratio. 6. The D2-like receptor agonist quinpirole reduced the S2/S1 ratio for [3H]dopamine release, an effect blocked by the antagonist AJ 76. The 5-HT1B receptor agonist CP 94253 during S2 reduced the S2/S1 ratio due to a reduction in evoked [3H]5-HT. This effect was blocked by the 5-HT1B receptor antagonist GR 55562. 7. The results demonstrate, for the first time, the functional presence of presynaptic alpha2-adrenoceptors, presynaptic 5-HT1B autoreceptors and presynaptic D2-like autoreceptors in broiler chicken hyperstriatal neurones in vitro.     

Surgical repair of femoral fractures in New World camelids: five cases (1996-2003)

Five New World camelids were admitted to the Western College of Veterinary Medicine between 1996 and 2003 for evaluation of femoral fractures. There were three alpacas and two llamas. Four of the animals were female and three were less than 3 months of age. Fracture configurations consisted of distal physeal fractures (three), a comminuted diaphyseal/metaphyseal fracture, and a transverse diaphyseal fracture. Fractures were diagnosed with a combination of physical examination and radiographs in all cases. All five fractures were repaired with internal fixation and three animals were discharged from the hospital with fractures that healed. One cria underwent successful internal fixation but died from pulmonary oedema during recovery from anaesthesia. Postoperative complications were rare and limited to inadequate fracture stability in one alpaca and prolonged recovery to weight bearing in another. One llama with a comminuted metaphyseal fracture, repaired with a 4.5 mm dynamic compression plate, subsequently had catastrophic failure of the bone 17 days after surgery. Overall the clients were pleased with the outcome of discharged animals. Although femoral fractures are considered rare, they pose a unique opportunity for the large animal veterinarian to successfully achieve fracture union with the aid of internal fixation.     

A free-choice intake of mineral blocks in beef cows during the grazing season and in winter

A herd of 25 beef cows were offered four types of mineral blocks (A, B, C, D) with a different Ca to P ratio (A 0.6:1, B 0.8:1, C 2:1 and D 3.6:1). The Mg content was identical in all blocks whereas the Na content was equal in B, C and D, while the Na content in A block was double. The mineral block intake was monitored for 1 year, which was broken down to the grazing period (Pa) and the winter period (Wi). The intake of pasture grass and winter forage was assessed and the feed chemical composition was analysed; based on the obtained values, a macroelement balance (for Ca, P, Mg, Na and K) was calculated.The results showed the forage diet met requirements for all the observed macroelements (except in Na). The macroelement intake from forage was considerably higher than from mineral blocks. There were significant seasonal (Pa versus Wi) differences in B and C blocks intake. The consumption of D block was relatively invariable all year round; block A was refused in both seasons though it contained the highest amount of highly deficient Na.It can be concluded that mineral content in forage meets beef cattle requirements for Ca, Mg and K, P content is just satisfactory, and Na content is insufficient. It appears that cows control, to some extent, the Ca to P ratio in their diet by choosing the appropriate type of mineral block. However, the preference or refusal of some types of mineral blocks regardless of the season suggests the influence of other factors affecting mineral block intake which were not the subject of our observation.     

Putative urinary pheromone of bulls involved with breeding performance of primiparous beef cows in a progestin-based estrous synchronization protocol

The objective of this study was to determine if factors associated with the biostimulatory effect of bulls alter breeding performance of primiparous, suckled beef cows using a progestin-based estrous synchronization protocol. We tested the hypotheses that the estrous synchronization response and AI pregnancy rates differ among cows exposed to bulls, continuously exposed to bull urine, and exposed to fence-line contact with bulls or cows not exposed to bulls or bull urine. Data were collected from 3 experiments performed over consecutive years. Cows were assigned to the following treatments: bull exposure (BE; n = 26) or no bull exposure (NB; n = 25) in Exp. 1, bull urine exposure (BUE; n = 19) or steer urine exposure (SUE; n = 19) in Exp. 2, and fence-line contact with bulls (BFL; n = 26) or no bull exposure (NB; n = 26) in Exp. 3. Synchronization protocols in each experiment included the use of a controlled internal drug release device (d -10), PGF(2alpha) (d -3), and GnRH and fixed-time AI (TAI; d 0). Cows that were observed in estrus by 60 h after PGF(2alpha) were inseminated 12 h later. Cows not observed in estrus by 60 h after PGF(2alpha) were TAI at 72 h and given GnRH (100 mug). Pregnancy was determined by ultrasonography 35 d after TAI. In Exp. 1, 2, and 3, cows were exposed directly to bulls, bull urine, or bull fence-line contact for 35, 64, and 42 d, respectively. Data were analyzed between treatments within each experiment. The proportion of estrous cycling cows did not differ between treatments at the beginning of each experiment; however, more (P < 0.05) BE and BFL cows were estrous cycling at the beginning of the estrous synchronization protocol than NB cows in Exp. 1 and 3. The proportion of cows that showed estrus and interval to estrus after PGF(2alpha) did not differ between treatments in Exp. 1 and 3. However, in Exp. 2, more BUE cows tended (P = 0.09) to have shorter intervals to estrus and to exhibit estrus after PGF(2alpha) than SUE cows. Overall, AI pregnancy rates were greater (P < 0.05) for BE and BUE cows than for NB and SUE cows in Exp. 1 and 2, respectively. There was no difference in AI pregnancy rates between BFL and NB cows in Exp. 3. The presence of bulls and exposure to bull urine appeared to improve breeding performance of primiparous beef cows using a progestin-based estrous synchronization protocol, whereas fence-line bull exposure was insufficient to cause this biostimulatory effect. We propose that a novel urinary pheromone of bulls may be responsible for the enhancement of fertility in the primiparous, postpartum cow.     

The use of a genetic-counselling program by Dutch breeders for four hereditary health problems in boxer dogs

Our group developed a genetic-counselling program for boxer-dog breeders in The Netherlands, using data for cryptorchidism (uni- and/or bilateral), epilepsy, knee-problems (including ligament rupture, fractured or ruptured meniscus, severe osteo-arthrosis of the knee, or a combination of these disorders), and schisis (including cheiloschisis, palatoschisis, or cheilopalatoschisis). We transformed the estimated breeding values (EBVs) into odds ratios (ORs), to enable the breeder to compare the risk for each of the traits for a certain dam-sire combination with the average population risk (set at 1). The goal of the study was to evaluate the use of our genetic-counselling program by Dutch breeders of boxer dogs. We asked breeders of the Dutch Boxer Club to send in an application form for genetic-counselling from June 1 to December 1, 2000. Breeders indicated on this application form three desirable sires for their dam (sire 1, sire 2, sire 3) in random order. On the basis of this information, a counselling report was produced which included ORs for the four diseases in litters of the dam-sire combinations indicated on the application form. Together with the counselling report, the breeders received an evaluation form. We received 129 application forms from 70 breeders, and collected 125 evaluations. Of these evaluations, 96 were informative about the influence of the counselling report on sire choice. The most-important criteria used by breeders to select sires were: the exterior characteristics (60%) and known progeny (52%). Although it was the first time breeders could make use of genetic-counselling, 32% of the breeders indicated that the genetic-counselling played a major role in their sire selection. Breeders expressed little difference in importance for the four genetic traits, but there was a tendency to consider epilepsy more than the others. Breeders hesitated to put long-term population interest above short-term personal interest. Nevertheless, the general conclusion of this study is that breeders acknowledge the value of counselling, especially to reduce the frequency of a threatening disease such as epilepsy.     

Effects of food withdrawal and transport on 5- to 10-day-old calves

The effects of food withdrawal for 30 hours and transport for up to 12 hours on 5- to 10-day-old calves were determined by monitoring plasma concentrations of glucose, beta-hydroxybutyrate, urea, lactate and creatine phosphokinase. In addition gamma-glutamyl transferase activity, packed cell volume, total plasma protein concentration, bodyweight and rectal temperature were recorded. Food withdrawal for 30 hours caused hypoglycemia but calves maintained normothermia and although their beta-hydroxybutyrate level increased indicating lipid mobilisation, the changes in blood urea level were minimal indicating low amino acid catabolism. When calves were transported for 12 hours in addition to having food withdrawn for 30 hours, they remained normoglycemic for six hours longer than non-transported calves, probably due to an effect of muscular activity during bracing against truck movements. Stocking calves at a lower density during transport apparently had a beneficial effect, as calves were able to lie down throughout the journey and therefore produced a similar metabolic profile to resting, non-transported animals. Transport and food withdrawal had no obvious effects on calf hydration. The results of this study suggest that food withdrawal for up to 30 hours and transport for up to 12 hours had no detrimental effects on the metabolism of healthy and clinically normal calves. With correct feeding regimes and transport protocols, welfare compromise in young, healthy calves being transported for up to 12 hours can be minimised when they are slaughtered within 30 hours of the start of transport.     

Plasma glucose-insulin relationship in chicken lines selected for high or low fasting glycaemia

1. Selected Fat or Lean chickens differ in their plasma glucose insulin relationship: in the fed or fasted state, Fat chickens have a lower glycaemia associated with normal or higher insulinaemia, depending upon the difference in glycaemia. 2. Conversely, chickens selected for low fasting glycaemia (LG) are fattier than their counterparts selected for high fasting glycaemia (HG), although the divergence in fat content is lower than in the Fat-Lean model. 3. The plasma glucose insulin relationship has been investigated in males of the HG and LG lines in the F4 and F5 generations. 4. A difference in glycaemia is suggested during embryonic development and was present at hatching and later on in the fasted or the fed state; insulinaemia did not differ. 5. During refeeding after an overnight fast, glycaemia differed between lines (except at intermediate times); cumulative food intake and insulinaemia were similar. 6. During a glucose tolerance test, glucose disposal rate and insulinaemia were rather similar. 7. Exogenous insulin exerted a very similar hypoglycaemic effect in both lines. 8. Other variables (body temperature, plasma concentrations of potassium and alpha NH2-non protein nitrogen) did not differ between HG and LG chickens. 9. In conclusion, HG and LG chickens do not exhibit any differences in glucose disposal rate, insulinaemia (in various nutritional conditions) or sensitivity to exogenous insulin, which contrasts with Fat or Lean chickens and may explain why HG and LG chickens have diverged to a lesser extent in fat content.     

Ultrasonographic assessment of maternal cardiac function and peripheral circulation during normal gestation in dogs

The aim of this study was to describe changes in cardiac morphology, systolic function and some peripheral hemodynamic parameters during normal pregnancy in dogs. Twenty healthy bitches, 10 pregnant (PG) and 10 non-pregnant controls (CG), were evaluated every 10 days using echocardiography from day 0 of the estrus cycle to parturition or to day 65 for the PG and CG groups, respectively. Systolic blood pressure (SBP) and uterine artery resistance index (RI) were also assessed. Throughout the study, the shortening fraction and cardiac output increased up to 30% vs. 5% (P<0.01) and 45% vs. 2% (P<0.01) in the PG and CG groups, respectively. In contrast, SBP and RI diminished up to 20% vs. 1% (P<0.01) and 29% vs. 0% (P<0.01) in the PG and CG groups, respectively. In conclusion, a decrease in afterload, an increase in cardiac output and cardiac hypertrophy appear to be the result of the hemodynamic modifications occurring during pregnancy in dogs.