Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV: Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 mu g oi the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of I-125-LH/CG-R added and (2) analysing sera for any chance in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (similar to 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED(50)) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED(50) of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) I-125-hCG binding to crude sheep luteal membrane (EC(50) of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence oi antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The: fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 130 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 mu g). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with hole LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Control of rabbits in arid Australia: destroying the drought refuge

Rabbits continued to infest Bulloo Downs in southwest Queensland even after rabbit haemorrhagic disease virus (RHDV) had effectively reduced rabbit populations to very low levels in most other arid parts of Australia. Control efforts for over 100 years have all appeared unable to stop rabbits causing damage to cattle production and native plants and animals in the area. In 2001 an experiment established to measure the benefit of rabbit control to biodiversity and cattle production showed warren ripping to cause an immediate reduction in rabbit activity. Three months after ripping there were still 98% fewer rabbits in ripped plots despite these plots being exposed to invasion from surrounding populations. The cost of ripping was high because of the high density of warrens and is prohibitive for a full-scale programme. Nevertheless, ripping warrens just in the rabbit’s drought refuge (2002 -2004) appears to have effectively controlled rabbits over the entire property. Following one good season rabbits still have not recovered where the drought refuge was effectively ripped. Destroying warrens in the areas where rabbits survived droughts achieved a reduction in rabbits of over 99% ompared to a similar area near Coongie Lakes in South Australia. Low rabbit numbers allowed cattle to continue to be run on the property even though the area experienced seven consecutive years with below average rainfall. It still remains to be seen whether rabbits can recover from this low population-base during a run of good seasons. If rabbit numbers remain suppressed after a run of good seasons then rabbit control by destruction of drought refuge could be repeated at Coongie Lakes and other drought refuge areas in the arid zone. Identification and treatment of areas similar to Bulloo Downs where rabbits survive drought may relieve a very large area of arid Australia from the damage caused by rabbits.

A method for mapping the distribution and density of rabbits and other vertebrate pests in Australia

The European wild rabbit has been considered Australia’s worst vertebrate pest and yet little effort appears to have gone into producing maps of rabbit distribution and density. Mapping the distribution and density of pests is an important step in effective management. A map is essential for estimating the extent of damage caused and for efficiently planning and monitoring the success of pest control operations. This paper describes the use of soil type and point data to prepare a map showing the distribution and density of rabbits in Australia. The potential for the method to be used for mapping other vertebrate pests is explored. The approach used to prepare the map is based on that used for rabbits in Queensland (Berman et al. 1998). An index of rabbit density was determined using the number of Spanish rabbit fleas released per square kilometre for each Soil Map Unit (Atlas of Australian Soils). Spanish rabbit fleas were released into active rabbit warrens at 1606 sites in the early 1990s as an additional vector for myxoma virus and the locations of the releases were recorded using a Global Positioning System (GPS). Releases were predominantly in arid areas but some fleas were released in south east Queensland and the New England Tablelands of New South Wales. The map produced appears to reflect well the distribution and density of rabbits, at least in the areas where Spanish fleas were released. Rabbit pellet counts conducted in 2007 at 54 sites across an area of south east South Australia, south eastern Queensland, and parts of New South Wales (New England Tablelands and south west) in soil Map Units where Spanish fleas were released, provided a preliminary means to ground truth the map. There was a good relationship between mean pellet count score and the index of abundance for soil Map Units. Rabbit pellet counts may allow extension of the map into other parts of Australia where there were no Spanish rabbit fleas released and where there may be no other consistent information on rabbit location and density. The recent Equine Influenza outbreak provided a further test of the value of this mapping method. The distribution and density of domestic horses were mapped to provide estimates of the number of horses in various regions. These estimates were close to the actual numbers of horses subsequently determined from vaccination records and registrations. The soil Map Units are not simply soil types they contain information on landuse and vegetation and the soil classification is relatively localised. These properties make this mapping method useful, not only for rabbits, but also for other species that are not so dependent on soil type for survival.

The value of having no wild rabbits in south-east Queensland

Rabbits released in Australia in 1859 spread to most areas of suitable habitat by 1910 causing great damage to the environment and primary industries. Measurement of damage is essential to justify spending money and utilising resources to remove rabbits. Damage to pasture and biodiversity may be irreversible and therefore difficult to measure without comparison with an area that has never suffered such damage. A rabbit proof fence completed in 1906 protected a large part of south east Queensland from rabbits. The Darling Downs Moreton Rabbit Board (DDMRB) continues to maintain the fence and keep the area relatively free of rabbits. This area is unique because it is highly suitable for rabbits and yet it has never ‘experienced’ the damage caused by plagues of uncontrolled rabbits. A study site was established where the DDMRB fence separates an area heavily used by rabbits (‘dirty side’) from an area that has never been infested by rabbits (‘clean side’). The number and location of all rabbit warrens and log piles were recorded. The absence of warrens from the ‘clean side’ shows clearly that the rabbit proof fence has prevented rabbits from establishing warren systems. The ‘dirty side’ is characterised by a high number of warrens, a high density of rabbits, fewer pasture species and low macropod activity. Future work will determine whether the rabbit populations are viable in the absence of rabbit warrens. We plan to radio collar rabbits on both sides of the fence to measure their survival rate. In selected warrens and log piles of varying degrees of complexity and size, rabbits will be trapped and information on reproduction and age structure will be collected. This will allow better targeting of the source of rabbits during control operations. Once the initial comparative analysis of the site has been completed, all rabbit warrens will be destroyed on the dirty side of the fence. After rabbits are removed from this area, monitoring will continue to determine if pasture and biodiversity on opposite sides of the fence begin to mirror each other.

Co-evolution of wild rabbits (Oryctolagus cuniculus) and RHDV: resistance and virulence

Rabbit Haemorrhagic Disease Virus (RHDV) was introduced to Australia in 1995 for the control of wild rabbits. Initial outbreaks greatly reduced rabbit numbers and the virus has continued to control rabbits to varying degrees in different parts of Australia. However, recent field evidence suggests that the virus may be becoming less effective in those areas that have previously experienced repeated epizootics causing high mortality. There are also reports of rabbits returning to pre-1995 density levels, Virus and host can be expected to co-evolve. The host will develop resistance to the virus with the virus subsequently changing to overcome that resistance. It has been 12 years since the release of RHDV and it is an opportune time to examine where the dynamic currently stands between RHDV and rabbits. Laboratory challenge tests have indicated that resistance to RHDV has developed to different degrees in populations throughout Australia. In one population a low dose (1:25 dilution) of Czech strain RHDV failed to infect a single susceptible rabbit, yet infected a low to high (up to 73%) percentage across other populations tested. Different selection pressures are present in these populations and will be driving the level of resistance being seen. The mechanisms and genetics behind the development of resistance are also important as the on-going use of RHDV as a control tool in the management of rabbits relies on our understanding of factors influencing the efficacy of the virus. Understanding how resistance has developed may provide clues on how best to use the virus to circumvent these mechanisms. Similarly, it will help in managing populations that have yet to develop high levels of resistance.

Should the 40-year-old practice of releasing virulent myxoma virus to control rabbits (Oryctolagus cuniculus) be continued?

Release of virulent myxoma virus has been a key component of rabbit-control operations in Queensland, Australia, since the 1960s but its use rests on anecdotal reports. During a routine operation to release virulent myxoma virus we found no evidence to support the continued regular use of the technique in south-west Queensland. Radio-tagged rabbits inoculated with virulent myxoma virus contracted the disease but failed to pass enough virus to other rabbits to spread the disease. Rabbits with clinical signs of myxomatosis that were shot were infected with field strain derived from the original laboratory strain released in 1950 rather than the virulent strain that has been released annually. There was no change in rabbit survival or abundance caused by the release. Nevertheless, the release of virulent virus may be useful against isolated pockets of rabbits mainly because field strains are less likely to be present. Such pockets are more common now that rabbit haemorrhagic disease virus is established in Queensland.

How can warren destruction by ripping control European wild rabbits (Oryctolagus cuniculus) on large properties in the Australian arid zone?

Context: For over 100 years, control efforts have been unable to stop rabbits causing damage to cattle production and native plants and animals on large properties in arid parts of Australia. Warren destruction by ripping has shown promise, but doubts about long-term success and the perceived expense of treating vast areas have led to this technique not being commonly used. Aims: This study measured the long-term reduction in rabbit activity and calculated the potential cost saving associated with treating just the areas where rabbits are believed to survive drought. Wealso considered whether ripping should be used in a full-scale rabbit control program on a property where rabbits have been exceptionally resilient to the influence of biological and other control measures. Methods: Rabbits were counted along spotlight transects before warrens were ripped and during the two years after ripping, in treated and untreated plots. Rabbit activity was recorded to determine the immediate and long-term impact of ripping, up to seven years after treatment. The costs of ripping warrens within different distances from drought refuge areas were calculated. Key results: Destroying rabbit warrens by ripping caused an immediate reduction in rabbit activity and there were still 98% fewer rabbits counted by spotlight in ripped plots five months after ripping. Seven years after ripping no active warrens were found in ripped plots, whereas 57% of warrens in unripped plots showed signs of rabbit activity. The cost of ripping only the areas where rabbits were likely to seek refuge from drought was calculated to be less than 4%of the cost of ripping all warrens on the property. Conclusions: Destroying rabbit warrens by ripping is a very effective way of reducing rabbit numbers on large properties in arid Australia. Ripping should commence in areas used by rabbits to survive drought. It is possible that no further ripping will be required. Implications: Strategic destruction of warrens in drought refuge areas could provide an alternative to biological control for managing rabbits on large properties in the Australian arid zone.

Rabbit haemorrhagic disease: are Australian rabbits (Oryctolagus cuniculus) evolving resistance to infection with Czech CAPM 351 RHDV?

Rabbit haemorrhagic disease is a major tool for the management of introduced, wild rabbits in Australia. However, new evidence suggests that rabbits may be developing resistance to the disease. Rabbits sourced from wild populations in central and southeastern Australia, and domestic rabbits for comparison, were experimentally challenged with a low 60 ID50 oral dose of commercially available Czech CAPM 351 virus - the original strain released in Australia. Levels of resistance to infection were generally higher than for unselected domestic rabbits and also differed (0-73% infection rates) between wild populations. Resistance was lower in populations from cooler, wetter regions and also low in arid regions with the highest resistance seen within zones of moderate rainfall. These findings suggest the external influences of non-pathogenic calicivirus in cooler, wetter areas and poor recruitment in arid populations may influence the development rate of resistance in Australia.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.