Sequential studies of skeletal calcium reserves and structural bone volume in a commercial layer flock.

1. Production-induced osteoporosis in caged laying hens is thought to represent a major constraint to continued genetic development. 2. The relationship between body weight, egg production, skeletal abnormalities characteristic of osteoporosis, femur calcium and bone histology was examined in a flock of ISA Brown layers from 16 to 68 weeks of age. 3. Experiment 2 examined a flock of Lohmann browns for skeletal abnormalities characteristic of osteoporosis at 45 weeks of age and the severity of abnormalities was then related to body weight and production between 18 and 45 weeks of age. 4. Average body weight declined in the ISA flock between 35 and 45 weeks of age, which correlated with a loss of skeletal calcium reserves (15% to 20%) and with the induction of osteoporosis. Between 42 and 68 weeks of age, birds were able to replenish femur calcium levels. 5. Birds in the Lohmann flock showing severe skeletal abnormalities at 45 weeks of age experienced weight loss between 27 and 31 weeks of age, which was associated with a decrease in egg production of 18%. After 35 weeks of age, egg production of these birds recovered to similar levels as unaffected or mildly affected birds. 6. It seems likely that better standardisation of the equilibrium between growth, skeletal reserves, food intake and egg production can reduce osteoporosis, as well as improving the productive potential of modern laying strains.

Changes in proteoglycan content of articular cartilage during avian degenerative joint disease.

OBJECTIVE: To determine the biochemical changes in articular cartilage composition associated with the development of avian degenerative joint disease (DJD) in ad libitum fed broiler fowl, in comparison to feed-restricted broilers and J-lin fowl (non-susceptible to DJD). METHODS: Articular cartilage from the distal tibiotarsus (DTT) was characterised up to age 180 days. Proteoglycan content was determined by uronic acid and sulphated glycosaminoglycan analysis, cellularity by assay for DNA content, and collagen content and crosslinking by hydroxyproline and pyridinoline analysis, respectively. RESULTS: Disease development was accompanied by increased hydration and proteoglycan content (particularly sulphated proteoglycans) and decreased cellularity, with no significant differences in either total collagen content or in mature collagen cross-linking. CONCLUSION: The biochemical features of avian DJD are similar to those observed in other animal models. This bipedal model is exceptional however since cartilage alterations occur spontaneously and in a load-dependent manner.

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.

In vivo development of Theileria annulata: major changes in efferent lymph following infection with sporozoites or allogeneic schizont-infected mononuclear cells.

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

Articular cartilage proteoglycan metabolism in avian degenerative joint disease: effects of strain selection and body weight.

The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.

Development of an experimental model of bacterial chondronecrosis with osteomyelitis in broilers following exposure to Staphylococcus aureus by aerosol, and inoculation with chicken anaemia and infectious bursal disease viruses.

A series of experiments was designed in an attempt to reproduce bacterial chondronecrosis with osteomyelitis in broiler chickens using a natural route of infection. Birds in isolators were exposed to a suspension of Staphylococcus aureus by aerosol or exposed to S. aureus and subsequently inoculated with chicken anaemia virus (CAV) alone, or with CAV and infectious bursal disease virus (IBDV). Subsequently, S. aureus was recovered and bacterial chondronecrosis with osteomyelitis was diagnosed, by histology, in the proximal end of the femur and/or tibiotarsus of lame birds exposed to S. aureus with and without CAV and IBDV infections. Birds fed 60% of the recommended feed intake for the breed developed a lower incidence of S. aureus infection and/or bacterial chondronecrosis (P < 0.05) than birds fed 100% of the recommended intake. A significantly lower incidence of S. aureus was recovered (P < 0.05) in birds simultaneously exposed to S. aureus and inoculated with CAV and IBDV at day 21, than in birds exposed to S. aureus at day 10, and inoculated with CAV and IBDV at day 21. With the exception of birds exposed to S. aureus at 1 day old, a higher incidence of bacterial chondronecrosis was diagnosed in birds exposed to S. aureus and inoculated with CAV and IBDV than in birds exposed to S. aureus alone. It is hypothesised that inoculation with CAV and IBDV at day 21 enhanced the development of bacterial chondronecrosis in birds exposed to S. aureus at day 10 and fed 100% of the recommended feed intake or ad libitum.

Study of leg weakness in two commercial broiler flocks.

The major causes of leg weakness/lameness were investigated in two male commercial broiler flocks. The numbers of dead and lame birds culled from the flocks each day were recorded by the flock managers. Forty-four lame birds and 22 sound birds were examined postmortem during a period of six weeks and the proximal and distal end of each femur, tibiotarsus and tarsometatarsus were examined histologically. Attempts were made to isolate bacteria and viruses from the proximal end of each femur. Blood samples were examined for antibodies to chicken anaemia virus (CAV), infectious bursal disease virus (IBDV) and Mycoplasma species. Bacterial chondronecrosis with osteomyelitis was identified in the proximal end of the femur of eight of the 44 lame birds, and in the proximal end of the tibiotarsus of a further bird (20.4 per cent). Gram-positive bacteria were present in all the lesions. Staphylococcus aureus was recovered from 62.5 per cent of the lesions confirmed by histology. Bacterial chondronecrosis associated with S aureus has thus been identified as an important cause of leg weakness in these commercial broilers. Lesions suggestive of the condition were visible macroscopically in only 11.1 per cent of the cases subsequently diagnosed by histology and bone histology is therefore required before a diagnosis can be excluded. Angular limb deformities (13.6 per cent) and spondylolisthesis (11.4 per cent) were the most common macroscopic lesions identified as causes of lameness. The overall incidence of tibial dyschondroplasia was similar in both the lame and sound broilers, but severe lesions were found only in lame birds (4.5 per cent).

Effects of body mass and genotype on avian degenerative joint disease pathology and articular cartilage proteoglycan distribution.

OBJECTIVE: The objective of this study was to investigate the role of body mass and genotype in the development of avian degenerative joint disease (DJD). METHODS: Layer strain and broiler strain fowl, fed either ad libitum or on a restricted diet, were kept under identical conditions for up to a year. At various time points cartilage samples were taken from the distal tibiotarsus (DTT), proximal tarsometatarsus, antitrochanter and proximal humerus. All samples were assessed for gross morphology and histopathology, and in some samples the cartilage proteoglycan distribution was investigated by Safranin O staining. RESULTS: Layer strain fowl did not develop DJD. Heavy ad libitum fed broiler strain fowl developed DJD earlier and more severely than lighter, feed restricted, broiler strain fowl. The articular surface of the DTT was worst affected by DJD. Safranin O staining of DTT samples (age 180 days) from the ad libitum fed broilers revealed variable proteoglycan distribution in the articular cartilage. Some areas were intensely stained throughout all zones, whereas other areas showed no staining in any zone. Age matched, non-diseased DTT samples from feed restricted broilers showed a more consistent staining pattern with little staining in the surface zone and more in the middle and deep zones. CONCLUSIONS: These data indicate that avian DJD is body mass mediated in broiler strain fowl, and that proteoglycan distribution is altered in diseased cartilage.

Estrogen and cancellous bone loss in the fowl.

Female birds model a type of woven bone prior to egg laying which is known as medullary bone. Medullary bone modeling is estrogen dependent and in the female fowl coincides with a decrease in cancellous bone volume. Medullary bone modeling was induced in male laying-strain fowl by the administration of estrogen and prevented in females by the administration of tamoxifen. In estrogen-treated males, medullary bone modeling was accompanied by cancellous bone loss; cancellous bone volume was significantly lower than in control males (P < 0.001). In females, the prevention of medullary bone modeling by tamoxifen treatment resulted in significantly higher cancellous bone volumes than in control females (P < 0.001). Estrogen therefore appears to play a role in cancellous bone loss in the fowl.

Gross, histological and histomorphometric features of the navicular bone and related structures in the horse.

Forelimb navicular bones and associated soft tissues were collected from 3 groups of horses and subjected to pathological examinations. The groups consisted of 38 horses with clinical navicular disease (ND) and 2 control groups, with no history of forelimb lameness, consisting of 25 age-matched mature horses (A-MC) and 9 immature horses (IC). Histological and histomorphometric studies were performed on tissue samples from 10 ND, 10 A-MC and 5 IC horses. Gross changes seen only in ND horses included: full thickness defects in the palmar surface fibrocartilage, palmar cortex erosion, medullary lysis, flexor digitorum profundus tendon (FDPT) surface fibrillation, FDPT core lesions and adhesions between the FDPT and navicular bone. Palmar surface partial thickness fibrocartilage loss and distal border fragmentation were seen with a significantly greater incidence in ND than in A-MC and not observed in IC. Remodelling of the proximal border, FDPT surface colouration, palmar surface fibrocartilage colouration and proximal border entheseous bone were identified in ND and A-MC but not in IC. Mid-ridge synovial fossae and horizontal depressions in the palmar surface were identified in all groups. Histologically palmar fibrocartilage thinning and loss were associated with reduced palmar fibrocartilage cell density and chondrocyte cluster formation. Palmar fibrocartilage fibrillation, palmar cortical bone defects, fibromyxoid stromal change in the medulla, medullary pseudocyst formation and entheseous new bone formation were all seen in ND. The adjacent FDPT showed fibrillation, tag formation and degeneration of the dorsal surface. Necrotic foci were also present within the body of the tendon. Although not always present, medullary bone pseudocysts, separate mineralised foci and most changes on the dorsal surface of the FDPT were specific to ND. Bone histomorphometric parameters were compared among groups. Cross-sectional area reduced from the sagittal ridge to the medial and lateral margins of each navicular bone. IC navicular bones had a smaller subchondral area, subchondral bone volume and a greater osteoid volume than in the AC, indicating that these differences were age-related. In ND the medullary area was decreased but the trabecular bone volume increased. The palmar subchondral area was increased but contained bone with an increased porosity and osteoid volume. Changes occurred from the medial to the lateral margins of the bone in horses with ND indicating remodelling of the bony elements throughout the bone in ND. The histological and histomorphometric changes in the navicular bone and palmar fibrocartilage were considered similar of those found in articular hyaline cartilage and subchondral bone in osteoarthritis.

Cloning differentially regulated genes from chondrocytes using agarose gel differential display.

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.

Bisphosphonates: a potential role in the prevention of osteoporosis in laying hens.

Osteoporosis in layers is associated with the modelling and remodelling of medullary bone. Cancellous bone volume (CBV) decreases initially during medullary bone modelling and continues to decrease during subsequent remodelling. In an attempt to maintain peak structural bone mass, the bisphosphonate, alendronate, was administered to pullets before medullary bone modelling. At point of lay CBV was significantly greater (P<0.01) in the alendronate group (17.59 per cent) than in controls (13.79 per cent), while medullary bone volume (MBV) was not significantly affected. After 20 weeks, CBV remained significantly higher (P<0.02) in the alendronate group (12.72 per cent) than in controls (9.80 per cent) and MBV was lower in the alendronate group than the control group. CBV was however reduced and MBV increased in both groups compared with values at point of lay. Alendronate therefore appeared to prevent the bone loss associated with medullary bone modelling but not that which occurs during remodelling.

A method for immunofluorescent localization of oestrogen receptors in bone sections from an egg-laying poultry strain.

Although oestrogen has profound skeletal effects in hens, the identity of its target cells in bone is still unclear. We wished to address this by indirect immunofluorescent detection of oestrogen receptors, using monoclonal antibodies, similar to our method for mammalian bone. Avian bone, however, is prone to autofluorescence at the excitation wavelength for fluorescein isothiocyanate, and non-specific binding of mammalian antibodies. We therefore improved receptor detection by comparing three commercially available monoclonal antibodies to the human oestrogen receptor. We found that the best identification of oestrogen target cells was produced by ID5 antibody diluted 1/20, with initial binding disclosed by Cy3trade mark-conjugated immunoglobin, which has similar fluorescence to rhodamine. Clear localisation of these cells was reliably obtained in sections of both receptor positive human breast tissue and hen oviduct. Preliminary observations showed that immunofluorescence in avian oviduct and undecalcified bone cryosections was stable after 6 weeks storage and of sufficient clarity for semiquantification. Thus, in hens aged 18 weeks (first ovarian follicle), osteoblasts and 38% of osteocytes were clearly immunofluorescent. After 8 to 10 weeks egg lay, receptor-positive osteocytes decreased in structural bone to 19%; cells adjacent to medullary bone and in marrow cavities were strongly immunofluorescent.

Degenerative joint disease in poultry--differences in composition and morphology of articular cartilage are associated with strain susceptibility.

The morphology and basic biochemical composition of articular cartilage from two strains of fowl were examined. Broiler breeder fowl are considered susceptible to degenerative joint disease (DJD); histological examination of one-year-old broiler breeders showed in some samples, articular cartilage thinning, fibrillation and chondrocyte cluster formation, features considered typical of DJD. Examination of similar samples from laying strain fowl showed only minor age-related changes such as some slight cartilage thinning and very mild fibrillation. The articular cartilage from the broiler breeder birds was significantly more hydrated with a higher uronic acid content than that of the laying strain birds. In addition, unloaded articular surfaces such as the proximal humerus had significantly higher amounts of uronic acid than the loaded cartilage surfaces of the proximal tarsometatarsus and the distal tibiotarsus; this suggested that the joint loading may have a role in any biochemical differences found between joints and between strains of fowl. These findings concur with other reports in mammals that showed increased hydration and uronic acid in association with early DJD and in models of osteoarthritis (OA). Thus, despite some differences between avian and mammalian articular cartilage, studies on avian DJD may give insights into mammalian disease.

An assessment of the efficacy of the Lixiscope for the detection of tibial dyschondroplasia.

The efficacy of Lixiscope examination in detecting tibial dyschondroplasia (TD) is assessed utilising a thorough post-mortem examination with good histological backup. Forty-seven per cent more broilers had TD at post-mortem examination than when examined solely by Lixiscope. TD lesions detected by Lixiscope examination had a mean score greater than 3. The additional lesions detected at post-mortem examination usually had a score of 1. This indicates that the Lixiscope examination failed to detect small lesions. The prevalence of dyschondroplastic lesions in the proximal tarsometatarsi indicates that this site may be of value to score in addition to the proximal tibiotarsus and use in selection. Disruption of the proliferative zone within some dyschondroplastic growth plates indicated that in a proportion of broilers there is a link between TD and rickets. The Lixiscope is an effective tool for detecting large TD lesions, but fails to detect smaller lesions. To minimize the susceptibility of genetic lines of broilers to TD it may be necessary to use progeny testing.

The development of lameness and bone deformity in the broiler following experimental infection with Mycoplasma gallisepticum or Mycoplasma synoviae.

Groups of eight 7-day-old Mycoplasma-free broiler chicks were inoculated with one of two strains of M. gallisepticum (MG/S6, MG/B31/85) or one of two strains of M. synoviae (MS/B31/88 and MS/B94/91) into the left hock joint. Controls were similarly inoculated with sterile mycoplasma broth. The birds were assessed at 2, 3, 4 and 5 weeks of age (1, 2, 3 and 4 weeks post-injection) for lameness and joint and limb lesions. At 5 weeks of age they were killed and examined for gross skeletal abnormalities and microscopic lesions of both hock joints. The in-contact controls showed no joint swelling or lameness. The mycoplasma did not apparently spread to cause gross pathology in the joints of in-contact control birds, or to other joints in a bird injected with mycoplasma. Each mycoplasma strain caused specific pathologies manifest as varying degrees of lameness, left hock joint swelling, bone deformity and differences in the severity of articular cartilage and chondrodystrophic growth plate pathology. Shortening and bowing of the right tibiotarsus in the birds injected with mycoplasma indicated unilateral increased limb loading contributing to bone deformity. Chondrodystrophy seen with three mycoplasmal strains may have contributed to bone deformity in these birds. Lameness appeared to be a consequence of either joint swelling and/or bone deformity resulting in mechanical dysfunction. The role of joint pain in the lameness was not determined.