Regulation of osteoarthritis by omega-3 (n-3) polyunsaturated fatty acids in a naturally occurring model of disease.

OBJECTIVE: To examine effects of high omega-3 (n-3) polyunsaturated fatty acid (PUFA) diets on development of osteoarthritis (OA) in a spontaneous guinea pig model, and to further characterise pathogenesis in this model. Modern diets low in n-3 PUFAs have been linked with increases in inflammatory disorders, possibly including OA. However, n-3 is also thought to increases bone density, which is a possible contributing factor in OA. Therefore we aim to determine the net influence of n-3 in disease development. METHOD: OA-prone Dunkin-Hartley (DH) Guinea pigs were compared with OA-resistant Bristol Strain-2s (BS2) each fed a standard or an n-3 diet from 10 to 30 weeks (10/group). We examined cartilage and subchondral bone pathology by histology, and biochemistry, including collagen cross-links, matrix metalloproteinases (MMPs), alkaline phosphatase, glycosaminoglycan (GAG), and denatured type II collagen. RESULTS: Dietary n-3 reduced disease in OA-prone animals. Most cartilage parameters were modified by n-3 diet towards those seen in the non-pathological BS2 strain - significantly active MMP-2, lysyl-pyridinoline and total collagen cross-links - the only exception being pro MMP-9 which was lower in the BS2, yet increased with n-3. GAG content was higher and denatured type II lower in the n-3 group. Subchondral bone parameters in the DH n-3 group also changed towards those seen in the non-pathological strain, significantly calcium:phosphate ratios and epiphyseal bone density. CONCLUSION: Dietary n-3 PUFA reduced OA in the prone strain, and most disease markers were modified towards those of the non-OA strain, though not all significantly so. Omega-3 did not increase markers of pathology in either strain.

A Necropsy Study of Disease and Comorbidity Trends in Morbidity and Mortality in the Koala (Phascolarctos cinereus) in South-East Queensland, Australia.

Koalas are an iconic Australian marsupial undergoing precipitous population reduction in South-East Queensland from complex interacting threats. To investigate the causes of death and the interaction of comorbidities with demography in South-East Queensland koalas, a large scale, high-throughput prospective necropsy survey was conducted spanning 2013-2016. During this period, 519 necropsies were conducted in 155 young/subadult koalas, 235 mature, 119 old koalas and 10 of unknown age. Similar numbers of males and females were assessed. Trauma and infectious disease at were the most common single diagnoses. However, comorbidity was frequent, including multicentric infection or infectious disease in combination with trauma or senescence. Female koalas had proportionally more reproductive chlamydiosis compared to males in which the ocular and urinary systems were more commonly affected. Comorbidity and disease were strongly associated with poor body condition, and trauma was associated with good body condition. Animals affected by motor vehicle trauma were often in better body condition than those affected by animal attack, tree fall or other causes of trauma. This study identified a higher frequency of infections and comorbidity then previously reported, confirming the complex nature of interacting threats to the koala population.

Effects of housing, parturition and diet change on the biochemistry and biomechanics of the support structures of the hoof of dairy heifers.

This study investigated effects of housing, diet and parturition on the biochemistry, biomechanics and pathology of feet of maiden, pregnant and lactating dairy heifers. Strength/laxity, laminar morphology, connective tissue (CT) biochemistry and sole lesions were assessed. Although no animals became clinically lame, severity of sole lesions was significantly greater in heifers housed in cubicles vs. straw yards, and in lactating/pregnant heifers vs. maidens. These effects were additive. Cubicle housing and parturition each increased CT metabolism (and were additive), and altered CT composition. Similarly, both impaired the biomechanical resilience of the hoof. There were no effects for diet. The results indicate that parturition/lactation causes non-inflammatory changes in CT that impair resilience of the feet to external stresses associated with poor housing. This "parturition effect" appears to be unrelated to change in diet and relatively brief, unless exacerbated by additional stresses of housing. Thus heifer lameness may be significantly reduced through short-term, low-cost improvements in husbandry around the time of calving.

Pulmonary adiaspiromycosis in critically endangered northern hairy-nosed wombats (Lasiorhinus krefftii).

BACKGROUND: The northern hairy-nosed wombat (Lasiorhinus krefftii) is critically endangered, with only 200 individuals remaining in the wild. Individuals are rarely available for detailed pathological assessment and identification of disease threats to individuals is critically important to species conservation. CASE REPORT: Two male northern hairy-nosed wombats, part of the Richard Underwood Nature Refuge population, were presented for necropsy, 5 months apart. They were found to have succumbed to adiaspiromycosis caused by the fungus Emmonsia parva. Pathological presentations were of severe pulmonary oedema and fibrosis, and pleuritis, respectively. Characteristic fungal adiaspores were noted on histopathological examination. The wombats had concurrent variably severe ectoparasite and endoparasite burdens. CONCLUSION: These are the first reported cases of adiaspiromycosis in northern hairy-nosed wombats and the organism was associated with significant pathological changes. The rarity and the logistical challenges of presenting northern hairy-nosed wombats for pathological assessment are a challenge to identifying disease threats in this critically endangered species.

Increased dietary protein in the second trimester of gestation increases live weight gain and carcass composition in weaner calves to 6 months of age.

Genetically similar nulliparous Polled Hereford heifers from a closed pedigree herd were used to evaluate the effects of dietary protein during the first and second trimester of gestation upon foetal, placental and postnatal growth. Heifers were randomly allocated into two groups at 35 days after artificial insemination (35 days post conception (dpc)) to a single bull and fed high (15.7% CP) or low (5.9% CP) protein in the first trimester (T1). At 90 dpc, half of each nutritional treatment group changed to a high- or low-protein diet for the second trimester until 180 dpc (T2). High protein intake in the second trimester increased birth weight in females (P=0.05), but there was no effect of treatment upon birth weight when taken over both sexes. Biparietal diameter was significantly increased by high protein in the second trimester with the effect being greater in the female (P=0.02), but also significant overall (P=0.05). Placental weight was positively correlated with birth weight, fibroblast volume and relative blood vessel volume (P<0.05). Placental fibroblast density was increased and trophoblast volume decreased in the high-protein first trimester treatment group (P<0.05). There was a trend for placental weight to be increased by high protein in the second trimester (P=0.06). Calves from heifers fed the high-protein treatment in the second trimester weighed significantly more on all occasions preweaning (at 1 month (P=0.0004), 2 months (P=0.006), 3 months (P=0.002), 4 months (P=0.01), 5 months (P=0.03), 6 months (P=0.001)), and grew at a faster rate over the 6-month period. By 6 months of age, the calves from heifers fed high nutrition in the second trimester weighed 33 kg heavier than those fed the low diet in the second trimester. These results suggest that dietary protein in early pregnancy alters the development of the bovine placenta and calf growth to weaning.

Differences between the thermal stabilities of the three triple-helical domains of type IX collagen.

Fibre-forming collagens in dilute solution show highly co-operative helix-coil transitions at temperatures that are remarkably close to the body temperature of the animal from which the collagen was extracted. This close correlation holds across animal Phyla and the transition temperatures, which range from 5 degrees C to 40 degrees C, are adjusted to suit by changing the primary structure, especially the concentration of the water-bridge-enhancing hydroxyproline residue. Fibril-forming collagens are thermally stabilised by fibrillogenesis, which causes a loss of random coil configurational entropy by intermolecular and intramolecular cross-linking and by spacial confinement of the molecule within the lattice of the fibre. But this mechanism cannot apply to the full length of the type IX collagen molecule, since its COL3 arm, according to current models, projects out from the stabilising influence of the type II fibre. In this paper we examine the thermal stability of the type IX collagen molecule and its three triple-helical domains, thereby demonstrating that the COL3 arm is much more stable than the rest of the molecule. At a scanning rate of 60 deg. C/h COL3 exhibited an unfolding endotherm with a tmax at 49.0 degrees C, well above body temperature. Corresponding peak maxima for COL1 and COL2 were seen at 40.6 degrees C and 39.6 degrees C, respectively. The sizes of the thermally labile units of COL1, COL2 and COL3, calculated from the measured activation enthalpies, were 24, 28 and 28 residues, respectively, much smaller than type I (65 residues) because of the relatively short lengths of triple helix to be unfolded. However, unlike type I collagen, no regions of the required size were found completely devoid of hydroxyproline. Consequently, the intrinsic stabilities of these thermally labile units were higher than that of type I with DeltaH updownarrow DeltaS updownarrow for COL1, COL2 and COL3 being, respectively, 385 K, 371 K and 384 K, contrasting with the much lower 349 K of type I collagen. We therefore speculate that the increased thermal stability of the thermally labile units was caused by the presence of the water-bridge-enhancing residue, hydroxyproline. Finally the stabilisation of type IX collagen tissue is considered and an alternative structural organisation of the type IX molecule on the type II fibre is proposed.

Mechanisms of maturation and ageing of collagen.

The deleterious age-related changes in collagen that manifest in the stiffening of the joints, the vascular system and the renal and retinal capillaries are primarily due to the intermolecular cross-linking of the collagen molecules within the tissues. The formation of cross-links was elegantly demonstrated by Verzar over 40 years ago but the nature and mechanisms are only now being unravelled. Cross-linking involves two different mechanisms, one a precise enzymically controlled cross-linking during development and maturation and the other an adventitious non-enzymic mechanism following maturation of the tissue. It is this additional non-enzymic cross-linking, known as glycation, involving reaction with glucose and subsequent oxidation products of the complex, that is the major cause of dysfunction of collagenous tissues in old age. The process is accelerated in diabetic subjects due to the higher levels of glucose. The effect of glycation on cell-matrix interactions is now being studied and may be shown to be an equally important aspect of ageing of collagen. An understanding of these mechanisms is now leading to the development of inhibitors of glycation and compounds capable of cleaving the cross-links, thus alleviating the devastating effects of ageing.

Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance.

Bone collagen cross-links are now widely used to assess bone resorption levels in many metabolic bone diseases. The post-translational modifications of bone and other mineralizing collagens are significantly different from those of other type I collagen matrices, a fact that has been exploited during recent advances in the development of biochemical markers of bone resorption. The enzymatic collagen cross-linking mechanism is based upon aldehyde formation from specific telopeptide lysine or hydroxylysine residues. The immature ketoimine cross-links in bone form via the condensation of a telopeptide aldehyde with a helical lysine or hydroxylysine. Subsequent maturation to the pyridinoline and pyrrole cross-links occur by further reaction of the ketoimines with telopeptide aldehydes. In mineralizing tissues, a relatively low level of lysyl hydroxylation results in low levels of hydroxylysyl pyridinoline, and the occurrence of the largely bone specific lysyl pyridinoline and pyrrolic cross-links. The collagen post-translational modifications appear to play an integral role in matrix mineralization. The matrix of the turkey tendon only mineralizes after a remodeling of the collagen and the subsequent formation of a modified matrix more typical of bone than tendon. Further, disturbances in the post-translational modification of collagen can also affect the mineralization density and crystal structure of the tissue. In addition to their use as a convenient measure of matrix degradation, collagen cross-links are of significant importance for the biomechanical integrity of bone. Recent studies of osteoporotic bone, for example, have demonstrated that subtle perturbations in the pattern of lysine hydroxylation result in changes in the cross-link profile. These alterations, specifically changes in the level of the pyrrolic cross-link, also correlate with the strength of the bone. Further research into the biochemistry of bone collagen cross-links may expand current understanding and their clinical application in metabolic bone disease. This review also demonstrates the potential for further study into this area to provide more subtle information into the mechanisms and etiology of disease and aging of mineralizing tissues.

Allogeneic bone marrow transplantation for adult leukaemia with soy bean lectin fractionated marrow.

A heterogeneous group of 11 consecutive patients with leukaemia have been transplanted successfully with allogeneic marrow depleted of T lymphocytes by soy bean lectin agglutination and neuraminidase-treated sheep erythrocyte rosetting. Effective depletion was achieved, leaving less than 1% of donor T lymphocytes. Despite the small numbers of nucleated cells infused (mean 0.14 X 10(8)/kg) there was only moderately delayed recovery of peripheral blood counts and no graft failures have occurred. Standard methotrexate prophylaxis against graft-versus-host disease (GVHD) was also employed in the first four transplants. Only one case of mild grade I (skin only) acute GVHD has occurred and there has been no chronic GVHD to date. The group of patients show an actuarial cumulative survival of 55% with two early infective deaths (days 42 and 44 post-transplant) and three late deaths, two with leukaemic relapse and the third with probable viral encephalitis. The longest survivor is now 1109 days post-transplant. This series indicates that lectin fractionation of donor marrow, previously employed mainly in children, can also be effective in minimizing GVHD in adults without endangering successful engraftment.

Bone marrow processing and cryopreservation.

Three different closed procedures for concentrating bone marrow progenitor cells prior to cryopreservation have been compared. These were by a manual double centrifugation method, a Hemonetics 30 cell separator and an Aminco Celltrifuge. The best results were achieved using the paediatric pheresis set on the Hemonetics model 30. Marrow was frozen in 120 ml aliquots in a programmed freezer with rapid cooling of the freezing chamber during the phase change from the liquid to the solid state. After freeze-thawing the average nucleated cell recovery was approximately 50% and the progenitor cell recovery 80%.

Treating osteoarthritis in practice--the TOP Study.

This study consisted of two nationwide surveys. The first involved 209 general practitioners (GPs) from 22 sampling points in England, Scotland and Wales, and the second involved 510 patients with osteoarthritis (OA) (aged 60 years and above), rheumatoid arthritis (RA) or unspecified arthritis, who had visited their GP for treatment. The results showed that OA has a significant negative impact on patients' quality of life; 79% of patients stated that their condition limited their quality of life. Only 15% of GPs reported prescribing a conventional non-steroidal anti-inflammatory drug (NSAID) as first-line treatment for patients with arthritis. More than half (57%) stated that they would prescribe a simple analgesic or a compound analgesic before trying an NSAID. Forty four per cent of GPs stated that their main therapeutic objective when prescribing an NSAID was to minimise GI side-effects, and therefore start at a low dose. This implies that many patients are likely to be receiving sub-optimal doses. Experiencing breakthrough pain and GI side-effects were common reasons cited for patients re-presenting to GPs. GPs reported their expectation to prescribe GI protectants to a fifth (21%) of their patients on NSAIDs and estimated one in six patients (17%) take OTC products to relieve GI symptoms. This research demonstrates that there is considerable dissatisfaction with currently used treatment regimens for osteoarthritis, from both the GP and patient perspectives.

Recurrent aortoduodenal fistula: a lesson in management.

Aortoenteric fistula (AEF) is a rare but often lethal complication of aortic reconstructive surgery. The diagnosis is often delayed due to the high percentage of false-negative using upper gastrointestinal series, angiography, and endoscopy. The diagnosis of AEF must be considered in any patient with a prosthetic graft and gastrointestinal bleeding. There are essentially two modalities of treatment: replacement of the graft or total removal of all prosthetic material and the construction of an extra anatomic bypass. A case report of a recurrent aortoduodenal fistula is presented that illustrated both delay in diagnosis and inappropriate treatment. We agree with the general trend in the literature that the appropriate management of this devastating complication is complete removal of the prosthetic graft, wide drainage, high-dose antibiotics, and extra anatomic bypass. Any deviation from these basic treatment steps - such as replacement of the graft - ignores the infectious component of this complication and can result in the devastating lethal complication seen in this patient. With appropriate suggested management this once uniformly fatal complication can be treated effectively, resulting in an acceptable morbidity and mortality.

Acute iron intoxication with intestinal infarction.

A fatal case of acute iron intoxication with intestinal infarction is reported, with emphasis on the acute gastrointestinal effects of iron ingestion. The patient demonstrated the progressive necrotizing effects of retained iron on the gastrointestinal tract. Although moderate amounts of iron may be irritating and may produce diarrhea, larger amounts may cause paralytic ileus. Localized concentrations of iron may then exert corrosive effects and cause segmental infarctions and perforations. Emphasis must be given to the purging of iron products from the bowel. Should surgery become necessary because of localized infarction or perforation, we believe that it is advisable to not only thoroughly irrigate the peritoneal cavity but also irrigate the lumen of the bowel so as to remove as much of the remaining iron as possible, lest further areas of necrosis and perforation develop because of continued corrosive effects.

The influence of age and breed on stallion semen.

This study reports on the variation in semen quality and in spermatozoal and behavioral characteristics of 168 stallions representing 9 breeds and ranging in age from 2 to 26 yr. Semen samples were collected into an artificial vagina and the number of mounts and urethral pulsations per semen sample were recorded. Semen characteristics were examined for total volume, gel-free volume, gel volume, color score, mass activity, nonmotile spermatozoa, dead spermatozoa, semen density, spermatozoa concentration, total number of spermatozoa and semen pH. Morphological characteristics of the spermatozoa included abnormal heads, abnormal mid-pieces, abaxial mid-pieces, protoplasmic droplets and abnormal tails. Sources of variation were evaluated and the overall means calculated by least-squares analyses of variance for nonorthogonal data. The significance of breed effects and between stallion variability were estimated using mixed-model procedures. All semen characteristics with the exception of color and urethral pulsations had significant variation due to age. Semen quality (gel-free volume, sperm concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3 yr of age and over 11 yr. Significant breed variation was apparent in most characteristics except for pH, semen color, abnormal midpieces and urethral pulsations. It is recommended that both the age and breed of stallion be taken into consideration when evaluating stallion semen.

Seasonal variation in the estrous cycle of mares in the subtropics.

Data on the estrous cycles and sexual receptivity scores of 7 maiden Australian Stock Horse mares were used to study seasonal variation from December until the following November. Mares were grazed in paddocks containing both native and improved tropical pasture species. The study was conducted in South-East Queensland (27 degrees 27' South latitude). There were no differences between either the sexual receptivity or the lengths of natural (Mean=7.5 days, SEM=0.4) and PGF-induced estruses (Mean=7.6 days, SEM=0.4). Plasma estrogens were not related to differences in sexual receptivity scores between mares. The lowest incidence of estrus occurred at the time of the winter solstice (June 22) in the Southern Hemisphere. Winter estruses (Mean=9.3 days, SEM=0.8) were longer than those of summer (Mean=6.6 days, SEM=0.5) and autumn (Mean=6.6 days, SEM=0.9). Approximately 30% of estruses were anovulatory. Most of these occurred in autumn and winter (P<0.05). There was considerable variation in the occurrence of anovulatory estrus between mares. There were no differences between the lengths of ovulatory and anovulatory estruses. Ovulatory estruses were significantly longer in spring than in summer (P<0.05). There were no seasonal differences between the lengths of anovulatory estruses. The length of diestrus (Mean=16.6 days, SEM=0.3) was not affected by mare or season. Only 3 of the 7 mares did not cycle during winter (anestrus), as confirmed by progesterone concentrations of less than 1 ng/ml. Finally, there was no evidence of mares having 2 breeding seasons per year in this study.

Preliminary study of ovarian activity in fillies treated with a GnRH vaccine.

OBJECTIVE: To investigate the effects of two doses (200 and 400 mg) of a water-soluble gonadotrophin-releasing hormone vaccine on the ovarian activity of 2-year-old fillies. DESIGN: A controlled vaccination dose rate experiment. ANIMALS: Six 2-year-old Australian Stock Horse fillies were randomly allocated to three treatment groups; unvaccinated controls, those receiving 200 mg of the vaccine and those receiving 400 mg of the vaccine. RESULTS: Ovarian activity of the treated fillies was suppressed at the peak of breeding season while that of untreated controls continued normally. The control fillies displayed oestrous activity and behaviour. Suppression of ovarian activity occurred for 25 and 30 weeks in the 200 and 400 mg groups, respectively. These differences were not significant. Ovarian activity ceased 2 to 3 weeks after primary vaccination. Antibody titres were low (330) until after the booster immunisation when they rapidly peaked at 22,000 and 28,000 in the 200 mg and 400 mg groups, respectively. Plasma progesterone concentrations of the treated fillies remained below 3.18 nmol/L while GnRH was suppressed. The vaccine had no significant effect on plasma androstenedione concentrations. Recovery from the effect of the vaccine was associated with development of ovarian follicles, normal oestrous behaviour and ovulation. Three of the four treated fillies and one of the controls conceived during the next breeding season and foaled normally. All the treated fillies conceived and produced normal foals in the following two breeding seasons. CONCLUSION: Both dose rates suppressed ovarian function and prevented oestrous behaviour. These effects were reversible and the subsequent fertility of the vaccinated fillies was normal.

Unusual ovarian activity in a mare preceding the development of an ovarian granulosa cell tumour.

An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a granulosa cell tumour; the left ovary was smaller than normal and non-functional. Surgical removal of the right ovary before increasing photoperiod resulted in a return to function of the left ovary and a pregnancy to frozen semen on the second cycle following removal. Figures showing concentrations of inhibin, progesterone, androstenedione, oestradiol and testosterone are presented for this entire period. Unusual ovarian activity in the mare might be a prelude to the development of a granulosa cell tumour.

Suppression of testicular function using two dose rates of a reversible water soluble gonadotrophin releasing hormone (GnRH) vaccine in colts.

OBJECTIVE: To investigate the effect of two dose rates (200 and 400 ng) of a gonadotrophin releasing hormone (GnRH) vaccine on testicular function. DESIGN: A vaccination dose rate experiment. PROCEDURE: Two injections were administered 4 weeks apart to six colts in each treatment group. To maintain immunosuppression until the end of the breeding season, a third injection was given if antibody titres fell below 1000. RESULTS: Effective antibody titres were present for 12 to 27 weeks. Testosterone concentrations decreased from 2.22 to 0.31 nmol/L 6 weeks after primary vaccination. Androstenedione concentrations decreased from 1.78 to 0.28 nmol/L 5 weeks after vaccination. Testosterone and androstenedione concentrations above 0.69 and 0.87 nmol/L were attained 31 to 43 weeks after vaccination. Mean scrotal widths and lengths decreased over 29 weeks from 9.2 cm and 9.7 cm to 6.7 cm and 7.6 cm. At surgical castration these dimensions were 10.1 cm and 11.0 cm. Mean semen characteristics before vaccination and after recovery were: gel-free volume 16.5 and 13.5 mL, sperm concentration 295.5 x 10(6) and 315.6 x 10(6)/mL, total sperm per ejaculate 4041 x 10(6) and 4657 x 10(6) and live normal spermatozoa 32% and 60%. Histologically, the testes showed active spermatogenesis. The mean testicular parenchyma weights for the 200 and 400 mg groups were 129.0 g and 109.8 g. Daily sperm production per testis and per gram of testis for the 200 and 400 mg groups were 3.7 x 10(8) and 2.8 x 10(6), and 2.3 x 10(8) and 2.0 x 10(6). CONCLUSIONS: Both dose rates suppressed testicular function. Data showed that the vaccine effects were reversible. Individual immune response was less varied in the 200 mg group. Further work is necessary to achieve a less variable response in the immunosuppression of testicular function.