Intra-articular injection of tumor necrosis factor-alpha in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation.

OBJECTIVE: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. DESIGN: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. RESULTS: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. CONCLUSION: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.

Lack of association between Leptospira spp. serovars Hardjobovis and Pomona and pregnancy and mid-term abortion in New Zealand farmed red deer.

This paper investigates Leptospira borgpeterseni serovar Hardjobovis and L. interrogans serovar Pomona as potential causes of sub-optimum pregnancy rates and mid-term abortion in farmed red deer. Rising two-year-old (R2, n = 22,130) and mixed-age (MA, n = 36,223) hinds from 87 and 71 herds, respectively, throughout New Zealand were ultrasound scanned early in gestation (Scan-1) and a sub-sample re-scanned (Scan-2) 55-89 days later and mid-term daily abortion rate calculated. A sub-sample of sera from pregnant and non-pregnant hinds at both scans, both with (case herds) and without aborted hinds was tested for Leptospira using the microscopic agglutination test with titre cut-point ≥1:48 as positive. At Scan-1, 44.3% of 661 and 4.6% of 647 hinds were sero-positive for Hardjobovis and Pomona, respectively. The geometric mean titre (GMT) for Pomona was higher in pregnant than non-pregnant MA hinds (p = 0.015) but not in R2 hinds. At Scan-2 in case herds, 40.3% of 2242 and 7.1% of 2243 hinds were sero-positive for Hardjobovis and Pomona, respectively. There was no association between Hardjobovis or Pomona serology and non-pregnancy (Scan-1) or mid-term abortion (Scan-2) at animal or herd level. In case herds, GMT was higher in non-aborted than aborted hinds for Hardjobovis (p = 0.018) in MA herds and for Pomona in R2 herds (p = 0.006). No uteri from hinds not pregnant or aborting at either scan, or not rearing a calf to weaning, and fetuses as available, were positive on PCR. Evidence is insufficient to confirm that Leptospira Hardjobovis and Pomona play a significant role in sup-optimum early pregnancy or mid-term abortion rates in deer.

Shape resonances of oriented molecules: ab initio theory and experiment on hydrocarbon molecules

We report ab initio calculations of the x-ray absorption cross section for the near edge x-ray absorption fine structure of C2H6, and C2H4, and C2H2 at the C K-edge, based on a full multiple scattering formalism. The angular dependence of the electric dipole transition in the calculations as well as the angular dependent experiments for the oriented molecules give a good opportunity to compare both. The resonance can be assigned to a sigma(*) shape resonance. The multiple scattering formalism and the experiment agree well and thereby support the existence of such features in the spectra.

Managing anthelmintic resistance: untreated adult ewes as a source of unselected parasites, and their role in reducing parasite populations.

AIMS: To test the hypotheses that when untreated adult ewes are rotationally grazed (follow behind) on pastures after lambs receiving routine anthelmintic treatments, the ewes can function as a source of unselected parasites in refugia, capable of slowing the development of anthelmintic resistance, and suppress the build-up of parasites resulting from the development of anthelmintic resistance. METHODS: Firstly, the potential of untreated adult ewes to slow the development of anthelmintic resistance, and to suppress parasite populations under differing levels of anthelmintic efficacy, was investigated using a simulation model. Secondly, a field trial with three replicates of each treatment compared two grazing systems (lambs only vs lambs followed by ewes) and two types of anthelmintic, viz albendazole (ALB), to which resistance was present (faecal nematode egg count reduction (FECR)=57-59%) and ivermectin plus levamisole (IL), to which resistance was absent (FECR=97-99%), in a factorial treatment structure. Parasite populations were monitored using faecal nematode egg counts (FEC), faecal larval cultures, pasture larval sampling, and slaughter of tracer lambs. Animal performance was measured using liveweight, dag score, body condition score, and fleece weights. RESULTS: Model simulations indicated that parasites cycling in the untreated ewes could slow the development of resistance being selected for by the anthelmintic treatments given to lambs and this could occur without a nett increase in larval numbers on pasture. Further, as worm control in the lambs declined with increasing levels of anthelmintic resistance the ewes increasingly functioned as nett removers of parasite larvae, effectively reducing parasite population size. In the field trial, untreated adult ewes contributed to pasture infestations of most parasite species, but not Nematodirus spp. Parasite species on pasture and infecting lambs changed when ewes were present, but larval populations on pasture in the autumn were no greater than when lambs grazed alone. In the presence of anthelmintic resistance, parasite populations were reduced when ewes grazed in rotation with lambs, implicating the ewes as nett removers of parasite challenge. CONCLUSIONS: Untreated adult ewes were a source of unselected genotypes, capable of slowing the development of anthelmintic resistance in most, but not all, parasite species. Further, the potential of adult ewes to remove from pasture more parasite larvae than they contribute through faecal contamination indicates a potentially useful role in suppressing parasite populations, particularly when worm control in lambs is less effective as a result of anthelmintic resistance.

Selective and on-demand drenching of lambs: impact on parasite populations and performance of lambs.

AIM: To determine whether drenching regimes for lambs by which a proportion (10%) of the heaviest animals was selectively left untreated, or animals are only drenched 'on demand' when faecal nematode egg counts (FEC) exceeded a threshold level, would result in measurable increases in parasite larval challenge in the autumn and/or decreases in the performance of lambs. METHODS: A replicated study compared three drenching strategies in which mobs of lambs (n=360 in total) received either: a five-drench preventive programme, administered to all animals (Treatment 1); a five-drench preventive programme, but the 10% heaviest animals left untreated each time (Treatment 2); or drench treatments administered only when FEC exceeded 500 eggs per gram of faeces (epg) (Treatment 3). After the five-drench programme, animals in Treatments 1 and 2 were treated according to FEC as for Treatment 3. A triple-combination drench containing ivermectin, oxfendazole and levamisole, administered orally, was used for all treatments. There were nine farmlets, allowing three replicates of each treatment, in a completely randomised design. Parasite infestations on pasture were measured in autumn by pasture plucks, and worm burdens were monitored in tracer lambs, while the performance of lambs was assessed by liveweight gains, fleece weights, and body condition and dag scores. RESULTS: Increased numbers of Haemonchus contortus and Trichostrongylus colubriformis larvae on pasture were found in the autumn on farmlets treating selectively or on-demand (Treatments 2 and 3). No differences were detected in other parasite species. Mean liveweight gains did not differ between treatments but some differences were detected between drenched and undrenched lambs in Treatment 2. Mean body condition and mean dag scores of lambs in Treatment 3 tended to be lower and higher, respectively, than those of lambs in Treatment 1; Treatment 2 was generally intermediate. CONCLUSIONS: Drenching strategies for lambs designed to slow the development of anthelmintic resistance, by increasing the pool of susceptible worms available to dilute resistant survivors after treatment, resulted in increased numbers of H. contortus and T. colubriformis but not other species of parasite on pasture. The increased parasite challenge to lambs in the autumn was associated with small production losses, which may be acceptable to farmers wishing to implement such strategies. It is clear that further work is required on the interaction between management practices and the population dynamics of parasites, especially with regard to creating pools of susceptible genotypes to slow the development of drench resistance.

Drenching adult ewes: implications of anthelmintic treatments pre- and post-lambing on the development of anthelmintic resistance.

AIMS: To test the hypothesis that peri-parturient anthelmintic treatment of adult ewes, either pre-lambing with a controlled- release capsule (CRC) or at tail-docking with a short-acting oral formulation, would increase the rate of development of anthelmintic resistance, as compared to not drenching ewes and giving an additional drench to lambs in the autumn. Also, to evaluate the potential of routinely leaving 15% of the heaviest lambs untreated when drenching, as a means of slowing the development of anthelmintic resistance. METHODS: A replicated farmlet trial was run from 1999- 2004. Eleven farmlets, each consisting of five paddocks, were initially seeded with Ostertagia (=Teladorsagia) circumcincta and Trichostrongylus colubriformis parasites, these being a mixture of albendazole-susceptible and -resistant isolates to yield a 96% reduction in faecal nematode egg count (FEC) on drenching. Four prescriptive drenching regimes were applied; Treatments 1-3 were replicated three times and Treatment 4 twice. Treatments were as follows. Treatment 1: Ewes were given an albendazole CRC pre-lambing, and any ewes exceeding 65 kg liveweight were given two capsules simultaneously; lambs were given a five-drench preventive programme of treatments, orally, of albendazole on Days 0, 21, 42, 70 and 98 after weaning. Treatment 2: Ewes were given a single oral treatment of albendazole at docking (2-3 weeks after lambing), and lambs were given the same five-drench preventive programme as in Treatment 1. Treatment 3: Ewes remained untreated, while lambs were given a six-drench preventive programme of treatments, orally, of albendazole on Days 0, 21, 42, 70, 98 and 126 after weaning. Treatment 4: Ewes remained untreated, while lambs were given the same six-drench preventive programme as in Treatment 3, but the heaviest 15% of lambs were left untreated each time. Albendazole-resistance status was measured at least twice-yearly, using faecal egg count reduction tests (FECRTs) and larval development assays (LDA). In addition, controlled slaughter of drenched and undrenched tracer lambs was undertaken in the last 3 years. RESULTS: Resistance to albendazole increased most rapidly in Treatment 1, as measured by FECRT and LDA results, and worm burdens in tracer lambs. In Treatment 2, resistance developed slower than in Treatment 1 but faster than in Treatments 3 and 4, as measured by LDA; resistance in Treatment 2 developed more quickly than in Treatment 4, as measured by FECRTs. There was no significant difference between Treatments 3 and 4, although this approached significance in Ostertagia spp, as measured by LDA. CONCLUSIONS: Anthelmintic treatments to adult ewes around lambing time are likely to be more selective for resistance than additional treatments administered to lambs in the autumn. Farmers wishing to slow the emergence of anthelmintic resistance on their farms should look to minimise the administration of peri-parturient treatment of ewes. A trend to slower development of resistance where a proportion of lambs were left untreated at each drench suggests further work on this aspect of management of resistance is warranted.

Distribution, prevalence and host associations of Hymenoptera parasitic on Calliphoridae occurring in flystrike in New Zealand.

Between 1920 and 1930, four species of parasitoid Hymenoptera (Tachinaephagus zealandicus, Alysia manducator, Nasonia vitripennis and Brachymeria ucalegon) were imported to New Zealand as an aid in the control of the blowflies causing myiasis in sheep (flystrike). Their long-term effects have never been investigated and the extent to which they were successful in establishing and enlarging their ranges has been given scant regard over the last 60 years. A long-term epidemiological study of flystrike in New Zealand between 1984 and 1996 obtained 4061 samples of blowfly larvae from flystruck sheep and carrion and overall 1.1% of these larval samples (most from the North Island) were found to be parasitized. Tachinaephagus zealandicus and A.manducator were present in field strikes, together with the endemic Aphaereta aotea recently found to be a parasite of calliphorids. The endemic species Phaenocarpa antipoda was found in association with carrion but not flystrike. Neither N.vitripennnis nor B.ucalegon were isolated from larvae in field strikes, the former because it parasitizes only pupae and the latter may be extinct as it has not been sighted since its introduction in 1919. It is concluded that if parasitoid wasps are able to locate and parasitize blowfly larvae on struck sheep, then their parasitism rate on more accessible larvae on carcases may be correspondingly greater. For this reason their scope for use in integrated management of flystrike could be increased by culturing and field release.

Relative efficacy of organophosphorus insecticides against susceptible and resistant strains of the strike blowfly Lucilia cuprina (Calliphoridae) in New Zealand sheep.

Four groups of five Romney lambs were treated by plunge dipping with one of four registered organophosphorus flystrike preventatives. Untreated lambs acted as controls. The sheep were challenged at weekly intervals with larval implants of organophosphate-susceptible and -resistant strains of Lucilia cuprina. All four treatments provided 19-21 weeks protection against susceptible larvae but chlorfenvinphos provided the longest protection (16-17 weeks), followed by propetamphos (15-16 weeks), dichlofenthion (10-13 weeks) and diazinon (9-13 weeks), against the resistant strain.

Measurements of photon interference X-ray absorption fine structure (piXAFS).

Experimental data are presented which demonstrate the existence of a fine structure in extended X-ray absorption spectra due to interference effects in the initial photon state (piXAFS). Interference occurs between the incident electromagnetic wave and its coherently scattered waves from neighboring atoms. Using fine platinum and tungsten powders as well as polycrystalline platinum foil, piXAFS was measured in high-precision absorption experiments at beamline X1 at HASYLAB/DESY over a wide energy range. piXAFS is observed below and above absorption-edge positions in both transmission and total-electron-yield detection. Based on experimental data it is shown that piXAFS is sensitive to geometric atomic structure. Fourier-transformed piXAFS data carry information, comparable with that of EXAFS, about the short-range-order structure of the sample. Sharp structures occur in piXAFS when a Bragg backscattering condition of the incident X-rays is fulfilled. They allow precise measurement of long-range-order structural information. Measured data are compared with simulations based on piXAFS theory. Although piXAFS structures are similarly observed in two detection techniques, the importance of scattering off the sample for the measurements needs to be investigated further. Disentangling piXAFS, multielectron photoexcitations and atomic XAFS in high-precision measurements close to absorption edges poses a challenge for future studies.

Germ-line transformation of the Australian sheep blowfly Lucilia cuprina.

The Australian sheep blowfly, Lucilia cuprina, is the most important economic insect pest for the sheep industries in Australia and New Zealand. piggyBac-mediated germ-line transformation of L. cuprina was achieved with a helper plasmid that had the Drosophila melanogaster hsp70 promoter controlling expression of the transposase and a piggyBac vector with an EGFP marker gene. Two transformant lines were obtained, at a frequency of approximately 1-2% per fertile G0. One of these lines has a single copy of the transgene, the other most likely has four copies. This is the first report of germ-line transformation of L. cuprina and is an important step towards the generation of engineered strains that would be suitable for male-only release eradication/suppression programmes.