Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Production benefits from pre- and post-lambing anthelmintic treatment of ewes on commercial farms in the southern North Island of New Zealand

Abstract

AIMS: To measure the magnitude and variability in production responses to anthelmintic treatments administered to adult ewes around lambing.

METHODS: Ewes carrying twin lambs, from sheep and beef farms (eight in Year 1 and six in Year 2) in the Wairarapa region of New Zealand, were enrolled in 14 trials (part of an experiment carried out on one farm in one year). Experiment 1 compared ewes treated 2–4?weeks pre-lambing with a controlled release capsule (CRC) containing abamectin, albendazole, Se and Co, to ewes injected pre-lambing with a long-acting Se plus vitamin B₁₂ product, and to untreated ewes. Experiment 2 included these treatments, plus a CRC administered at pregnancy scanning. Experiment 3 included the same treatments as Experiment 1, plus administration of a CRC containing albendazole, Se and Co, injectable moxidectin or oral derquantel plus abamectin, all administered pre-lambing, or oral derquantel plus abamectin administered 4–6?weeks after lambing. Variables compared were ewe liveweight at weaning and pre-mating, lamb liveweight at weaning, total weight of lamb weaned per ewe and ewe dag score at weaning.

RESULTS: Ewes treated with a CRC pre-lambing were heavier than untreated ewes (mean 3.2?kg) at weaning in 12/14 trials, and pre-mating (mean 2.8?kg) in 9/14 trials (p<0.001). Compared with mineral-treated ewes the mean difference was 2.8?kg pre-lambing (9/14 trials) and 1.7?kg pre-weaning (6/14 trials). Lambs reared by treated ewes were heavier (mean 1.55?kg) at weaning in 6/14 trials (p<0.001), but there was no effect of CRC treatment on total weight of lambs weaned per ewe (p=0.507). Variation in weight of lamb weaned per ewe was largely explained by differences in lamb survival from birth to weaning (p<0.001), with no effect of CRC treatment (p>0.65).

Treatment of ewes with a CRC at pregnancy scanning was neither better nor worse than a pre-lambing treatment (p=0.065).

There was no difference in the response from treatment with either of the two CRC or moxidectin. Treatment with short-acting oral anthelmintics resulted in no consistent benefit.

CONCLUSIONS: Anthelmintic treatments administered to ewes around lambing resulted in variable responses between farms and years, which in some trials were negative for some variables, and some of the variability was due to the mineral component of the CRC. The widespread perception amongst farmers and veterinarians that anthelmintic treatment of ewes around lambing will always result in positive benefits is not supported.     

A general planner for agro-ecosystem models

In agro-ecosystem simulation models involving a farm, management is usually controlled through some combination of calendar- and logic-based rules. This approach has been quite successful but despite their apparent naturalness and simplicity, rules do present some difficulties in use and implementation. Even relatively simple rule sets can become quite large and difficult to follow, and they are based on the current state of the system rather than anticipating the likely future state of the system. Systems based on alternative approaches may have the capability to improve on the performance of rule-based systems and here we describe the implementation of such an alternative, called the general planner for agro-ecosystem models (GPAM), and discuss how the GPAM makes decisions in the presence of complex interactions.The GPAM works by constructing a decision tree of all possible decisions up to some defined point in the future, assessing which pathway through the decision tree leads to the best outcome, and then passing set of decisions defining that pathway to the simulation model to implement. To test its capabilities, the GPAM was instructed to control a grazing rotation in a very simple agro-ecosystem model. Tests were conducted to examine the reaction of the GPAM's performance to a range of parameters, including how far forward in time it looked when optimising the rotation length, how often it revised the rotation length plan, and how often it was allowed to change the rotation length. In all of these tests the GPAM reacted as expected and, without being provided with any prior knowledge, decided to implement the long winter rotation lengths normally used by farm managers in year-round grazing systems to make best use of limited pasture. The highest levels of animal production were obtained when the GPAM was able to make swift changes in the rotation length, when it looked further into the future when optimising the rotation length, and when it re-planned often.Initial testing indicated that the GPAM showed promise as a new way of emulating the manager in simulation models. More work is needed to assess the performance and potential of the GPAM when it is provided with imperfect or biased information on the likely future states and to allow the GPAM to manage multi-objective systems, such as when balancing production and environmental goals.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Evaluating commercially available rodenticide baits for invasive Gambian giant pouched rats (Cricetomys gambianus)

Gambian giant pouched rats (Cricetomys gambianus) are native to Africa, but they are popular in the pet industry in the United States. They were reservoir hosts during a monkeypox outbreak in the Midwestern United States in 2003. A free-ranging population became established on Grassy Key in the Florida Keys, apparently because of a release by a pet breeder. These rodents could cause significant damage to agricultural crops should they reach the mainland. Research under controlled conditions was needed to identify effective rodenticides for Grassy Key or other cases where an invasion of Gambian rats might occur. We tested 2 formulations of diphacinone baits and 1 formulation each of brodifacoum, zinc phosphide, bromethalin, and chlorophacinone baits with captive Gambian rats in multiple-choice food trials. Both the brodifacoum and zinc phosphide rodenticide baits were highly effective (100% mortality). Also, brodifacoum and zinc phosphide treatments performed similar to the Environmental Protection Agency’s standard for toxicants of (i.e., 90% mortality in laboratory trials). The chlorophacinone, diphacinone, and bromethalin baits did not appear to be very effective at killing Gambian rats (≤50% mortality) in our study. Effective tools to combat Gambian giant pouched rats have been identified in a laboratory trial. Further field testing of commercially available brodifacoum and zinc phosphide baits may prove useful for controlling the potentially invading Gambian rats.     

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2‐deoxy‐d ‐glucose, DOG). Furthermore, treatment with DOG also led to a dose‐dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.     

Metabolic and physiological effects of adrenoceptor agonists and antagonists in the horse.

In the horse the effect of the adrenergenic agonists adrenaline, phenylephrine and salbutamol on haematocrit, plasma free fatty acid, glycerol and lactate levels were investigated. Effects on heart rate, sweating and muscle tremor were also studied. The effects of administration of the adrenoceptor antagonists propranolol, metoprolol, H35/25 and acepromazine on adrenaline-induced changes were examined. The results obtained with these agonists and antagonists suggest that the lipolysis and hyperglycaemia are mediated via beta-adrenoceptors. It appears that both beta1 and beta2 subtypes are involved. Muscle glycogenolysis, muscle tremor and sweating were mediated via beta2-adrenoceptors. Although salbutamol caused an elevation in haematocrit the other results support the alpha-mediation of adrenaline induced increases in haematocrit.     

Ultrastructural variations in the sweat glands of anhidrotic horses

The ultrastructure of sweat glands from the skin of free sweating horses was compared with that of glands from anhidrotic cases. Evidence of atrophied and abnormal sweat glands in the anhidrotic horses indicates that the condition involves progressive failure of the glandular mechanism of sweat production.     

Treatment of virulent footrot with lincomycin and spectinomycin

A mixture of lincomycin and spectinomycin was investigated as a treatment for footrot in sheep. In a controlled clinical trial 92.5% of acute and chronic cases of virulent footrot were cured following a single intramuscular injection of a mixture containing 50 mg lincomycin and 100 mg spectinomycin/ml at a dose rate of 1 ml/10 kg bodyweight. No improvement in clinical response was observed in groups of sheep treated on 3 successive days with this dose rate nor in another group treated once at a dose rate 1 ml/3.3 kg bodyweight. Cure effectiveness of each of the 3 treatment groups relative to untreated controls was 89%, 95% and 95%. Efficacy of lincomycin/spectinomycin was compared with that of penicillin/streptomycin in the treatment of footrot on 2 farms in south western New South Wales. Assessments made 14 to 17 d after treatment showed that on one farm all 122 ewes treated with lincomycin/spectinomycin had recovered while 170 of 175 ewes treated with penicillin/streptomycin recovered in the same period. On the second farm 87 of 90 ewes treated with lincomycin/spectinomycin recovered, compared with 184 of 190 sheep in the same flock treated with penicillin/streptomycin. Supportive footbathing did not seem to improve the clinical response in either treatment group and the paring done was sufficient only to establish diagnosis and to remove grossly overgrown horn.