Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

BACKGROUND Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. METHODOLOGY/PRINCIPAL FINDINGS Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. CONCLUSIONS There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.

Establishment and characterisation of an open mini-thoracotomy surgical approach to an ovine thoracic spine fusion model

Background A large animal model is required for assessment of minimally invasive, tissue engineering based approaches to thoracic spine fusion, with relevance to deformity correction surgery for human adolescent idiopathic scoliosis. Here we develop a novel open mini–thoracotomy approach in an ovine model of thoracic interbody fusion which allows assessment of various fusion constructs, with a focus on novel, tissue engineering based interventions. Methods The open mini-thoracotomy surgical approach was developed through a series of mock surgeries, and then applied in a live sheep study. Customized scaffolds were manufactured to conform with intervertebral disc space clearances required of the study. Twelve male Merino sheep aged 4 to 6 years and weighing 35 – 45 kg underwent the abovementioned procedure and were divided into two groups of six sheep at survival timelines of 6 and 12 months. Each sheep underwent a 3-level discectomy (T6/7, T8/9 and T10/11) with randomly allocated implantation of a different graft substitute at each of the three levels; (i) polycaprolactone (PCL) based scaffold plus 0.54μg rhBMP-2, (ii) PCL-based scaffold alone or (iii) autograft. The sheep were closely monitored post- operatively for signs of pain (i.e. gait abnormalities/ teeth gnawing/ social isolation). Fusion assessments were conducted post-sacrifice using Computed Tomography and hard-tissue histology. All scientific work was undertaken in accordance with the study protocol has been approved by the Institute's committee on animal research. Results. All twelve sheep were successfully operated on and reached the allotted survival timelines, thereby demonstrating the feasibility of the surgical procedure and post-operative care. There were no significant complications and during the post-operative period the animals did not exhibit marked signs of distress according to the described assessment criteria. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL-based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluation of the respective groups. Conclusion. This novel open mini-thoracotomy surgical approach to the ovine thoracic spine represents a safe surgical method which can reproducibly form the platform for research into various spine tissue engineered constructs (TEC) and their fusion promoting properties.

A comprehensive ovine model of blood transfusion

Background The growing awareness of transfusion-associated morbidity and mortality necessitates investigations into the underlying mechanisms. Small animals have been the dominant transfusion model but have associated limitations. This study aimed to develop a comprehensive large animal (ovine) model of transfusion encompassing: blood collection, processing and storage, compatibility testing right through to post-transfusion outcomes. Materials and methods Two units of blood were collected from each of 12 adult male Merino sheep and processed into 24 ovine-packed red blood cell (PRBC) units. Baseline haematological parameters of ovine blood and PRBC cells were analysed. Biochemical changes in ovine PRBCs were characterized during the 42-day storage period. Immunological compatibility of the blood was confirmed with sera from potential recipient sheep, using a saline and albumin agglutination cross-match. Following confirmation of compatibility, each recipient sheep (n = 12) was transfused with two units of ovine PRBC. Results Procedures for collecting, processing, cross-matching and transfusing ovine blood were established. Although ovine red blood cells are smaller and higher in number, their mean cell haemoglobin concentration is similar to human red blood cells. Ovine PRBC showed improved storage properties in saline–adenine–glucose–mannitol (SAG-M) compared with previous human PRBC studies. Seventy-six compatibility tests were performed and 17·1% were incompatible. Only cross-match compatible ovine PRBC were transfused and no adverse reactions were observed. Conclusion These findings demonstrate the utility of the ovine model for future blood transfusion studies and highlight the importance of compatibility testing in animal models involving homologous transfusions.

Genetic Parameters for Production Traits in Queensland Merinos

Genetic and phenotypic parameters are presented for production traits, greasy fleece weight (GFW), yield (YLD), clean fleece weight (CFW), average fibre diameter (DIAM) and liveweight (LWT), in 15 month old medium Peppin Merino sheep at Longreach and Julia Creek, Queensland. Heritabilities for GFW, YLD, CFW, DIAM and LWT were respectively 0.35, 0.62, 0.34, 0.74, and 0.37 for Longreach and 0.23, 0.52, 0.20, 0.67 and 0.56 for Julia Creek. Most estimates were consistent with other reported values. AAABG 13th Conference; Proceedings of the Association for the Advancement of Animal Breeding and Genetics.

Ex Vivo Model for Percutaneous Vertebroplasty

The testing of novel biomaterials for percutaneous vertebroplasty depends on suitable animal models. The aim of this study was to develop ex vivo a reproducible and feasible model of percutaneous vertebroplasty, for ulterior application in vivo. A large animal model was used (Merino sheep), due to its translational properties. Vertebroplasty was performed under tactile and fluoroscopic control, through a bilateral modified parapedicular access in lumbar vertebrae (n=12). Care was taken in order to avoid disruption of the vertebral foramen. The average defect volume was 1234±240 mm3. This mean volume ensures practical defects to test novel injectable biomaterials. 6 vertebrae were injected with a commercial cement (Cerament®, Bone Support, Sweden). Adequate defect filling was observed in all vertebrae. All vertebrae were assessed by microCT, prior to and post defect creation and after biomaterial injection. All vertebrae were mechanical tested. No mechanical failure was observed under loads higher than the physiological. Ultimately, this model is considered suitable for pre-clinical in vivo studies, mimicking clinical application.

An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5 alpha and EQ1

Aims: To examine Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 for known pathogenic mechanisms. Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d postinfection. Conclusions: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. Significance and Impact of the Study: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

Critical evaluation of cashmere nutrition experiments and suggestions for the design and conduct of successful experiments

Cashmere fibre production is an order of magnitude less than fibre production of Merino sheep or Angora goats and is more difficult to measure. Based on a comparison between cashmere experiments reporting responses to nutrition and those reporting no response, 13 design and management characteristics were identified that are related to the ability of experiments to discriminate among treatments. Methods must be adopted to reduce the variance in cashmere production within treatments, by using sufficient. animals per treatment, having enough replication to provide plenty of degrees of freedom to reduce error terms in analysis, and using pre-experimental cashmere production attributes as co-variants in analysis. It is preferable to use more productive and older goats, and goats that are used to handling, and to the conditions and feed to be used. Nutrition treatments need to produce different live weight growth curves and an appropriate control is needed such as live weight maintenance. As the raw cashmere fleece is composed primarily of hair and other contaminants, careful attention is required to measure, sample and test cashmere. Cashmere growth experiments should start by midsummer and last for at least four and preferably six months. These requirements make it more difficult for many university students to plan, undertake and complete long-term cashmere nutrition experiments without considerable management support.

Relationship of body condition score, live weight, stocking rate and grazing system to the mortality of Angora goats from hypothermia and their use in the assessment of welfare risks

Body condition scoring is widely used for sheep and cattle but the practice is included in only one Code of Practice for the welfare of goats in Australia. There is no published scientific evidence to support or defend its use in the assessment of welfare risks to farmed goats.

PROCEDURE: The significance of stocking rate, grazing system, body condition score (CS) and live weight were investigated in explaining the risk of mortality of individual and flocks of grazing Angora goats from hypothermia following a severe weather event in April. This event occurred 5 weeks after shearing the goats. Angora goats and Saxon Merino sheep were grazed alone, or mixed together in equal numbers at each of three stocking rates.

RESULTS: There was no mortality amongst Angora goats provided they grazed at the lowest stocking rate even when their CS was < or = 2.0. Mortality in flocks of Angora goats was most related to the CS reached during the preceding 2 months. For flocks of Angora goats there was no mortality at CS > or = 2.5 and mortality increased sharply at mean CS < 2.0. For individual Angora goats, mortality increased as CS declined and stocking rate and grazing combinations were additive in effect on mortality. Grazing with sheep increased mortality of Angora goats at higher stocking rates. The individual goat mortality rate was not dependent on individual plot effects suggesting that these results are applicable widely. Live weight loss was not related to mortality rates of goats once CS had been accounted for.
CONCLUSION: It was concluded that CS and stocking rate were highly significant determinants of welfare risk in Angora goats.

A review of cashmere nutrition experiments with suggestions for improving their design and conduct

The scientific literature contains divergent views about the effects of nutrition on cashmere. The consequences of ignoring nutrition will be an increase in the number of goats suffering lower production, increased welfare risks and premature mortality. This review evaluated published reports to identify current knowledge and best practice in regard to the design and management of cashmere nutrition experiments. The ability of experiments to distinguish between treatments was evaluated based on their statistical evidence. Many experiments had serious deficiencies in their design, conduct and reporting. Six of 16 papers did not provide statistical information that would enable a reader to verify differences between treatments. For most experiments to detect nutrition affects at P < 0.05, they required a difference between treatments of 0.2–0.8 μm in cashmere mean fibre diameter and 15–42 g in clean cashmere production. Government Research Institutes research was characterised by more experienced authors conducting longer (P < 0.05) and larger (P < 0.05) experiments than those conducted by Universities. Much of the “debate” regarding the affects of nutrition on cashmere production arises from the misinterpretation of both experiments that did not detect statistically significant effects and of experiments that did detect statistically significant effects. Based on a comparison between experiments reporting responses to nutrition with those reporting no response, 13 design and management features were identified that are related to the ability of experiments to detect significant treatment affects. Methods must be adopted to reduce the variance in cashmere production within treatments, by using sufficient animals per treatment, and having replication to provide sufficient degrees of freedom to reduce error terms in analysis. The power of experimental designs should be evaluated before experiments commence. Cashmere production records from a previous full production year could be used as co-variants during statistical analyses but this requires that potential experimental goats are managed in one flock, without variations resulting from different grazing, reproduction or other management for a year prior to an experiment. It is preferable to use more productive and older goats, and goats that are used to handling, and to the conditions and feed to be used. Allocation of animals to treatments must take into account live weight. Nutrition treatments need to be sufficiently different to produce different growth curves. An appropriate control is needed such as live weight maintenance. Evidence of both nutrition intake and growth curves must be published with cashmere production data so the claims made can be verified by the actual responses. As cashmere production is an order of magnitude less than fibre production of Merino sheep or Angora goats and is more difficult to measure, the requirements for measurement, sampling and testing cashmere fleeces are summarised. The use of mid side cashmere patches to determine cashmere growth and quality is seriously biased and must be avoided, preferably by shearing goats prior to and at the end of experiments. In order to obtain higher fleece growth responses and improve the ability of experiments to detect treatment effects it is preferable to start cashmere growth experiments by midsummer and conduct experiments for at least 4 months. These requirements make it difficult for many university students to plan, undertake and complete long-term cashmere nutrition experiments without considerable management support. It is not possible for experiments to disprove the Null hypothesis regarding the effects of nutrition on cashmere production as they can only report a failure to detect treatment effects. Researchers and journals need to be more rigorous in providing statistical information including: degrees of freedom for error terms, treatment variances, standard error of differences or similar to enable readers to compare treatment effects. Publications that do not provide sufficient statistical information should be disregarded from future discussions. Claims that an experiment shows no responses to nutrition should be subject to rigorous examination using the issues identified in this review.

Fibre production by beef cows

Cattle grow and shed fibre which assists them adapt to seasonal changes in the environment. In the absence of cattle fibre production data for southern Australia, Angus, Hereford, Simmental and Limousin cows and crosses between these breeds grazing perennial pastures at Hamilton, Victoria were sampled in late winter. The fibre-growing area on the sides of cattle was measured, fibre sampled at the mid-side site and the sampling area determined. Fibre was tested for fibre diameter distribution, clean washing yield and fibre length measured. Cows were 3-7 years of age, liveweights were 412-712 kg and the mean fibre-growing area was 2.2 m². This produced an average 682 g of total fibre (range 3461-175 g). The mean fibre diameter of all fibres was 51.7 μm (range 43-62 μm) and 18% of fibres were 36 μm (range 6-39%). The clean washing yield was 92.4% (range 87.4-95.8%). Fibre length averaged 21 mm. Increasing the age, liveweight and condition score of cows and increasing weight of clean fibre were associated with significant increases in mean fibre diameter. Breed of cattle did not affect fibre production (P > 0.1) but did affect mean fibre diameter (P < 0.05). The quantity of fibre production indicates potential for low value textile production. The high level of total fibre production, twice that of an earlier report, and fibre shedding from cattle suggests that white fibre-producing animals such as Merino sheep, Angora and cashmere goats and alpaca should avoid using cattle-handling facilities, particularly in the month before shearing.

Sunlight exposure caused yellowing and increased mineral content in wool

This study determines how levels of various trace metals in wool and the colour of the fibre change as a result of sunlight exposure and treatment with chelating compounds during wool growth. Twenty-four yearling Merino sheep were clipped on the shoulders and rumps and fitted with sheep coats modified with transparent patches. Patches over the shoulder wool (one per sheep) were either polyethylene (PE) that transmits ultraviolet light or polyvinyl chloride (PVC) that excludes ultraviolet light. The rump wool on each sheep was treated either with a copper chelator treatment (kojic acid or methyl gentisate in aqueous alcohol) or aqueous alcohol only. For 12 of the sheep the rumps were exposed to sunlight through PE patches while rump wool on the other sheep was covered by the sheep coat. Wool was harvested after 11 weeks’ growth with yellowness (Y-Z) and individual mineral contents measured using the same clean wool sample. Sunlight exposure through PE patches caused a mean increase in Y-Z to 9.1 (shoulder) or 9.5–10.1 (rump) from a base level of 7.1–7.2 (shoulder) or 7.0–7.6 (rump) in wool protected by the sheep coat. In contrast, there was no significant change in Y-Z for the PVC patch (shoulder). Therefore, it appears that ultraviolet light damage caused the increased Y-Z. Most of the trace metals analysed increased in the shoulder wool exposed to sunlight but the paired differences for PVC were lower than PE. It appears that changes in fibre caused by sunlight exposure (especially ultraviolet light) facilitate adsorption of minerals from the environment, including the animal’s own suint. Application of the chelating compounds to the rump wool caused pronounced yellowing of the wool with Y-Z increase being most pronounced for kojic acid. Copper levels in the wool were reduced by kojic acid and methyl gentisate while calcium levels were increased by kojic acid and reduced by methyl gentisate. It is not clear from these findings whether minerals and copper in particular contribute to yellowing of wool. However, the different effects of sunlight and chelation on mineral contents in wool shown may well relate to alternative mechanisms of discoloration (i.e. photoyellowing versus bacterial).

The allometric relationship between clean mohair growth and the fleece-free liveweight of Angora goats is affected by liveweight change

Clean fleece weight (CFWt) is affected by liveweight and change in liveweight in Merino sheep, Angora and cashmere goats. However, how these relationships progress as animals age has not been elucidated. Measurements were made over 12 shearing periods on a population of Angora goats representing the current range and diversity of genetic origins including South African, Texan and interbred admixtures of these and Australian sources. Records of breed, sire, dam, date of birth, dam age, birthweight, birth parity, weaning weight, liveweight, fleece growth and fleece quality were taken for does and castrated males (wethers) (n = 267 animals). Fleece-free liveweights (FFLwt) were determined for each goat at shearing time by subtracting the greasy fleece weight from the liveweight recorded immediately before shearing. The average of the FFLwt at the start of the period and the FFLWt at the end of the period was calculated (AvFFLwt). Liveweight change (LwtCh) was the change in FFLwt over the period between shearings. A restricted maximum likelihood model was developed for CFWt, after log10 transformation, which allowed the observations of the same animal at different ages to be correlated in an unstructured manner. A simple way of describing the results is: CFWt = κ (AvFFLwt)β, where κ is a parameter that can vary in a systematic way with shearing age, shearing treatment and LwtCh; and β is an allometric coefficient that only varies with LwtCh. CFWt was proportional to FFLwt0.67 but only when liveweight was lost at the rate of 5–10 kg during a shearing interval of 6 months. The allometric coefficient declined to 0.3 as LwtCh increased from 10 kg loss to 20 kg gain during a shearing interval. A consequence is that, within an age group of Angora goats, the largest animals will be the least efficient in converting improved nutrition to mohair.

The value of visual fleece assessment in addition to objective measurements in identifying Angora goats of greater clean mohair production

Visual assessment of the fleece of Merino sheep is an accepted method to aid genetic improvement but there is little evidence to support the use of visual assessment for improving mohair production. This paper examines the extent that visual traits, including staple length, character (staple crimp), staple definition, tippiness, style and staple entanglement, are related to clean fleece weight in animals of similar live weight and mean fibre diameter (MFD) from the same flock. Measurements were made over 9 shearing periods on a population of castrated Angora males (wethers) goats representing the current range and diversity of genetic origins in Australia, including South African, Texan and interbred admixtures of these and Australian sources (these different genetic origins are defined as Breed in this work). Data on genetic origin, sire, dam, lifetime characteristics (date of birth, dam age, birth weight, birth parity (single or twin), weaning weight), live weight, fleece growth and visual fleece attributes were recorded. A restricted maximum likelihood (REML) model was developed to relate clean fleece weight with age, MFD, average fleece-free live weight, lifetime characteristics and visual fleece attributes. There were separate linear responses of clean fleece weight to MFD and staple length for each age group, a quadratic response to the square root of average fleece-free live weight, an effect of sire breed and linear responses to dam age, staple definition score and character. Depending on age at shearing, the increase in clean fleece weight was between about 50 and 80. g for each increase of 1. μm in MFD. At similar MFD, clean fleece weight was generally greater at summer shearings compared with winter shearings. There was a strong increase in clean fleece weight with average fleece-free live weight up to around 50. kg but little response in clean fleece weight for animals larger than 50. kg. There was some evidence of a smaller increase in clean fleece weight as the age of dam increased. There was an effect of Breed in the model but this effect disappeared when a random sire effect was included in the model. There was a positive response to staple length at some age groups but the response did not differ from zero in other age groups. This response varied from negligible to about 70. g per 1. cm increase in staple length. Clean fleece weight increased about 40. g per unit increase in staple definition score and increased about 30. g for every 4 units increase in the number of staple crimps. There was no evidence that clean fleece weight was affected by staple style, staple tip score or staple entanglement score or lifetime factors such as birth weight, date of birth, birth parity, or weaning weight. The results show that using a combination of measuring MFD and visually assessing the fleece for staple length, staple definition and crimps can help identify the most profitable Angora goats. In this process, the objective measurement of MFD appears essential. Visual assessment will provide some extra benefit in identifying these animals above that provided by measuring MFD alone. Animal size should be considered by mohair producers when identifying more productive mohair producing animals. © 2014 Elsevier B.V.