Osteosarcoma with widespread metastasis in a 1-year-old crossbred rabbit.

A 14-month-old female spayed, small crossbred rabbit presented for assessment of a small, hard subcutaneous nodule in the right axilla. Serum biochemistry showed markedly increased serum ALP activity. A whole-body CT revealed an aggressive, monostotic osteolytic, and productive lesion within the left alveolar process of the maxilla, with erosion of the alveolar bone and secondary premolar depression. Innumerable metastatic osseous masses were present throughout the body, including cerebral, pulmonary, hepatic, subcutaneous, and skeletal muscular metastases. Postmortem findings confirmed widespread, metastatic osteosarcoma, with the primary lesion within the left maxilla.

Infrared Spectroscopy of Synovial Fluid Shows Accuracy as an Early Biomarker in an Equine Model of Traumatic Osteoarthritis.

Osteoarthritis is a leading cause of lameness and joint disease in horses. A simple, economical, and accurate diagnostic test is required for routine screening for OA. This study aimed to evaluate infrared (IR)-based synovial fluid biomarker profiling to detect early changes associated with a traumatically induced model of equine carpal osteoarthritis (OA). Unilateral carpal OA was induced arthroscopically in 9 of 17 healthy thoroughbred fillies; the remainder served as Sham-operated controls. The median age of both groups was 2 years. Synovial fluid (SF) was obtained before surgical induction of OA (Day 0) and weekly until Day 63. IR absorbance spectra were acquired from dried SF films. Following spectral pre-processing, predictive models using random forests were used to differentiate OA, Sham, and Control samples. The accuracy for distinguishing between OA and any other joint group was 80%. The classification accuracy by sampling day was 87%. For paired classification tasks, the accuracies by joint were 75% for OA vs. OA Control and 70% for OA vs. Sham. The accuracy for separating horses by group (OA vs. Sham) was 68%. In conclusion, SF IR spectroscopy accurately discriminates traumatically induced OA joints from controls.

A frameshift-deletion mutation in Reelin causes cerebellar hypoplasia in White Swiss Shepherd dogs.

Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic origins, with Mendelian-effect mutations described in several mammalian species. Here, we describe a genetic investigation of cerebellar hypoplasia in White Swiss Shepherd dogs, where two affected puppies were identified from a litter with a recent common ancestor on both sides of their pedigree. Whole genome sequencing was conducted for 10 dogs in this family, and filtering of these data based on a recessive transmission hypothesis highlighted five protein-altering candidate variants - including a frameshift-deletion of the Reelin (RELN) gene (p.Val947*). Given the status of RELN as a gene responsible for cerebellar hypoplasia in humans, sheep and mice, these data strongly suggest the loss-of-function variant as underlying these effects. This variant has not been found in other dog breeds nor in a cohort of European White Swiss Shepherds, suggesting a recent mutation event. This finding will support the genotyping of a more diverse sample of dogs, and should aid future management of the harmful allele through optimised mating schemes.

Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses.

Biomarkers for osteoarthritis (OA) in horses have been extensively investigated, but translation into clinical use has been limited due to cost, limited sensitivity, and practicality. Identifying novel biomarkers that overcome these limitations could facilitate early diagnosis and therapy. This study aimed to compare the concentrations of synovial fluid (SF) and plasma cell-free DNA (cfDNA) over time in control horses with those with induced carpal OA. Following an established model, unilateral carpal OA was induced in 9 of 17 healthy Thoroughbred fillies, while the remainder were sham-operated controls. Synovial fluid and plasma samples were obtained before induction of OA (Day 0) and weekly thereafter until Day 63, and cfDNA concentrations were determined using fluorometry. The SF cfDNA concentrations were significantly higher for OA joints than for sham-operated joints on Days 28 (median 1430 µg/L and 631 µg/L, respectively, p = 0.017) and 63 (median 1537 µg/L and 606 µg/L, respectively, p = 0.021). There were no significant differences in plasma cfDNA between the OA and the sham groups after induction of carpal OA. Plasma cfDNA measurement is not sufficiently sensitive for diagnostic purposes in this induced model of OA. Synovial fluid cfDNA measurement may be used as a biomarker to monitor early disease progression in horses with OA.

Bone quality changes as measured by Raman and FTIR spectroscopy in primiparous cows with humeral fracture from New Zealand.

The occurrence of spontaneous humeral fractures in primiparous dairy cows from New Zealand prompted the study of bone material from affected cows to further characterize this condition and to outline a likely pathogenesis. Previous studies indicate that these cows developed osteoporosis due to periods of suboptimal bone formation followed by increased bone resorption during the period of lactation complicated by copper deficiency. We hypothesized that there are significant differences in the chemical composition/bone quality in bones from cows with spontaneous humeral fracture compared to cows without humeral fractures. In this study, Raman and Fourier transform infrared spectroscopy band ratios were, for the first time, measured, calculated, and compared in bone samples from 67 primiparous dairy cows that suffered a spontaneous fracture of the humerus and 14 age-matched post-calving cows without humeral fractures. Affected bone showed a significantly reduced mineral/matrix ratio, increased bone remodeling, newer bone tissue with lower mineralization and, lower carbonate substitution, and reduced crystallinity. As such, is likely that these have detrimentally impacted bone quality and strength in affected cows.

Osteoporosis is the cause of spontaneous humeral fracture in dairy cows from New Zealand.

Outbreaks of humeral fractures in dairy cows have been reported in New Zealand for several years. Gross, histologic, and histomorphometric findings in the humerus from primiparous cows with spontaneous humeral fracture were compared to age-matched control cows. Affected cows had a complete nonarticular spiral fracture of the humerus. Histologically affected humeri had a thicker growth plate with abnormal architecture, thinner cortex with increased abnormal resorption, increased resorption in the distal humerus, decreased trabecular density, abnormal trabecular architecture, presence of growth arrest lines and woven bone formation. Histomorphometry showed reduction in bone volume, trabecular perimeter, and trabecular width. Cows grazed on fodder beet had thicker growth plates with an abnormal appearance compared with cows grazed on pasture, and cows with low/marginal liver copper concentration had more resorption cavities in the distal humerus and thinner cortical bone compared with cows with adequate liver copper concentration. Decreased trabecular density (OR = 249.5), abnormal cortical resorption (OR = 54.2), presence of woven bone formation in the proximal metaphysis (OR = 37.2), and the number of resorption cavities in the distal humerus were significantly associated with a high probability of fracture. Ribs had enlargement of the costochondral junction with fractures in different stages of healing. Histology of the ribs revealed abnormal growth plate appearance, presence of fracture lines, callus tissue, fibrosis, and microfractures. Cows with humeral fracture have osteoporosis due to decreased bone formation and increased bone resorption, likely associated with inadequate feed quality and perhaps copper deficiency leading to a reduction in bone strength and fracture.

Undetectable vitamin D3 in equine skin irradiated with ultraviolet light.

Vitamin D requirements for most animals are expected to be fulfilled through daily exposure of the skin to solar ultraviolet B radiation. The synthesis of vitamin D3 in skin depends on different factors including melanin pigmentation, the amount of UVB radiation reaching the skin, type of clothing/hair coat, latitude and altitude, season, and time of day. Alternatively vitamin D2 may be obtained from UVB irradiated pasture species. Recent studies have shown that in unsupplemented grazing horses 25-hydroxyvitamin D2 is the predominant form of vitamin D in plasma, and that 25OHD3 is undetectable suggesting horses may rely on diet to obtain vitamin D. In order to mimic the natural environment of skin to sunlight exposure, five equine and two ovine devitalized skin samples were irradiated with 5 J/cm2 of UVB light followed by measurement of 7-dehydrocholesterol (7-DHC) and vitamin D3 concentrations using reverse-phase high pressure liquid chromatography (HPLC). HPLC revealed the presence of 7-DHC in the skin of both horses and sheep. Vitamin D3 was undetectable in both ovine and equine skin prior to irradiation, but after irradiation with UVB light, ovine skin showed an increase in vitamin D3 concentration (mean 0.16 ± 0.07 µg/g), whereas vitamin D3 was undetectable in equine skin. These results provide additional evidence that horses make negligible quantities of vitamin D3 in their skin after exposure to UVB light and may therefore rely on their diet as a primary source of vitamin D.

Novel Assessment of Collagen and Its Crosslink Content in the Humerus from Primiparous Dairy Cows with Spontaneous Humeral Fractures Due to Osteoporosis from New Zealand.

Numerous cases of spontaneous humeral fracture in primiparous dairy cows from New Zealand have prompted the study of the condition to establish probable causes or risk factors associated with the condition. Previous studies identified inadequate protein-calorie malnutrition as an important contributory factor. Earlier case studies also reported that ~50% of cows have low liver and/or serum copper concentration at the time of humeral fracture. Because copper is so closely associated with the formation of collagen cross-links, the aim of this study was to compare collagen and collagen crosslink content in the humerus from primiparous cows with and without humeral fractures and to determine the role of copper in the occurrence of these fractures. Humeri were collected from cows with and without humeral fractures, ground, and the collagen and collagen cross-link content measured using high-performance liquid chromatography. Collagen content was significantly higher in the humeri of cows without humeral fractures, while total collagen crosslink content was significantly higher in the humerus of cows with humeral fractures. These results indicate other factor/s (e.g., protein-calorie undernutrition) might be more important than the copper status in the occurrence of humeral fractures in dairy cows in New Zealand.

Multimodal Blockade of the Renin-Angiotensin System Is Safe and Is a Potential Cancer Treatment for Cats.

The role of the renin-angiotensin system (RAS) in cancer growth and progression is well recognized in humans. However, studies on RAS inhibition with a single agent have not shown consistent anticancer effects, potentially due to the neoplastic cells utilizing alternative pathways for RAS activation. To achieve more complete RAS inhibition, multimodal therapy with several medications that simultaneously block multiple steps in the RAS has been developed for use in humans. In the present study, the safety of multimodal RAS inhibition using atenolol, benazepril, metformin, curcumin, and meloxicam was assessed in six cats with squamous cell carcinomas. Cats were treated for 8 weeks, with blood pressure measured and blood sampled five times during the treatment period. None of the cats developed hypotension, azotemia, or increased serum liver enzyme concentrations. The packed cell volume of one cat decreased to just below the reference range during treatment. One cat was reported to have increased vomiting, although this occurred infrequently. One cat was withdrawn from the study due to difficulties administering the medications, and another cat died of an unrelated cause. Two cats were euthanatized during the study period due to cancer progression. Two cats completed the 8-week study period. One was subsequently euthanized due to cancer progression while the other cat is still alive 32 weeks after entering the study and is still receiving the multimodal blockade of the RAS. This is the first evaluation of multimodal blockade of the RAS in veterinary species. The study showed that the treatment is safe, with only mild adverse effects observed in two treated cats. Due to the small number of cats, the efficacy of treatment could not be evaluated. However, evidence from human studies suggests that a multimodal blockade of RAS could be a safe and cost-effective treatment option for cancer in cats.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV-67, bla SHV-11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

Telangiectatic Osteosarcoma of the Tibiotarsus in a North Island Brown Kiwi (Apteryx mantelli).

A North Island brown kiwi (Apteryx mantelli) with lameness and weight loss had a telangiectatic osteosarcoma. The left proximal tibiotarsus had bony lysis with multiple blood-filled spaces separated by thick fibrous septa and neoplastic mesenchymal cells producing osteoid (5-bromo-4-chloro-3'-indolyl phosphate/nitro blue tetrazolium positive on cytology). No metastases were noted on necropsy.

Pathology of the peripheral neuropathy Charcot-Marie-Tooth disease type 4H in Holstein Friesian cattle with a splice site mutation in FGD4.

Charcot-Marie-Tooth disease (CMT) is a hereditary sensory and motor peripheral neuropathy that is one of the most common inherited neurological diseases of humans and may be caused by mutations in a number of different genes. The subtype Charcot-Marie-Tooth disease type 4H (CMT4H) is caused by homozygous mutations in the FGD4 (FYVE, RhoGEF, and PH domain-containing 4) gene. A previous genome-wide association study involving 130,783 dairy cows found 6 novel variants, one of which was a homozygous splice site mutation in the FGD4 gene. Descendants of carriers were genotyped to identify 9 homozygous Holstein Friesian calves that were raised to maturity, of which 5 were euthanized and sampled for histopathology and electron microscopy at 2 and 2.5 years of age. Three control Holstein Friesian animals were raised with the calves and euthanized at the same time points. No macroscopic lesions consistent with CMT4H were seen at necropsy. Microscopically, peripheral nerves were hypercellular due to hyperplasia of S100-positive Schwann cells, and there was onion bulb formation, axonal degeneration with demyelination, and increased thickness of the endoneurium. On electron microscopy, decreased axonal density, onion bulb formations, myelin outfoldings, and increased numbers of mitochondria were present. These changes are consistent with those described in mouse models and humans with CMT4H, making these cattle a potential large animal model for CMT.

An inherited night blindness in Wiltshire sheep.

Twelve cases of adult-onset blindness were identified in a flock of 130 polled Wiltshire sheep in New Zealand over a 3-year period. Affected sheep developed night blindness between 2 and 3 years of age, which progressed to complete blindness by 4 to 5 years of age. Fundic examination findings included progressive tapetal hyperreflectivity and attenuation of retinal blood vessels. Histologically, the retinas had a selective loss of rod photoreceptors with initial preservation of cone photoreceptors. Retinal degeneration was not accompanied by any other ocular or central nervous system abnormalities, and pedigree analysis suggested an inherited basis for the disease. Mating an affected Wiltshire ram to 2 affected Wiltshire ewes resulted in 6 progeny that all developed retinal degeneration by 2 years of age, while mating of the same affected ram to 6 unaffected ewes resulted in 8 unaffected progeny, consistent with autosomal recessive inheritance. Homozygosity mapping of 5 affected Wiltshire sheep and 1 unaffected Wiltshire sheep using an OvineSNP50 Genotyping BeadChip revealed an identical-by-descent region on chromosome 5, but none of the genes within this region were considered plausible candidate genes. Whole-genome sequencing of 2 affected sheep did not reveal any significant mutations in any of the genes associated with retinitis pigmentosa in humans or progressive retinal atrophy in dogs. Inherited progressive retinal degeneration affecting rod photoreceptors has not been previously reported in sheep, but this disease has several similarities to inherited retinal dystrophies in other species.

Growth and Bone Development in the Horse: When Is a Horse Skeletally Mature?

Within the lay literature, and social media in particular, there is often debate about the age at which a horse should be started and introduced to racing or sport. To optimize the welfare and longevity of horses in racing and sport, it is important to match exercise with musculoskeletal development and the ability of the musculoskeletal system to respond to loading. The justification for not exercising horses at a certain age is often in contrast to the scientific literature and framed, with incorrect generalizations, with human growth. This review provides a relative comparison of the growth and development of the horse to the descriptors used to define growth and development in humans. Measures of physeal closure and somatic growth demonstrate that the horse completes the equivalent of rapid infant growth by weaning (4-6 months old). At approximately 11 months old, the horse completes the equivalent of the childhood phase of growth and enters puberty. At 2 years old, the horse has achieved most measures of maturity used within the human literature, including the plateauing of vertical height, closure of growth plates, and adult ratios of back length:wither height and limb length:wither height. These data support the hypothesis that the horse evolved to be a precocious cursorial grazer and is capable of athletic activity, and use in sport, relatively early in life.

Non-additive association analysis using proxy phenotypes identifies novel cattle syndromes.

Mammalian species carry ~100 loss-of-function variants per individual1,2, where ~1-5 of these impact essential genes and cause embryonic lethality or severe disease when homozygous3. The functions of the remainder are more difficult to resolve, although the assumption is that these variants impact fitness in less manifest ways. Here we report one of the largest sequence-resolution screens of cattle to date, targeting discovery and validation of non-additive effects in 130,725 animals. We highlight six novel recessive loci with impacts generally exceeding the largest-effect variants identified from additive genome-wide association studies, presenting analogs of human diseases and hitherto-unrecognized disorders. These loci present compelling missense (PLCD4, MTRF1 and DPF2), premature stop (MUS81) and splice-disrupting (GALNT2 and FGD4) mutations, together explaining substantial proportions of inbreeding depression. These results demonstrate that the frequency distribution of deleterious alleles segregating in selected species can afford sufficient power to directly map novel disorders, presenting selection opportunities to minimize the incidence of genetic disease.

The Effect of Sex and Age on Bone Morphology and Strength in the Metacarpus and Humerus in Beef-Cross-Dairy Cattle.

In cattle, limited data have been reported about the relationship between live weight, bone size, and strength and how this relationship can be altered by factors such as sex and age. The aim of this study was to describe the relationship of peripheral quantitative computed tomography (pQCT)-derived parameters of bone strength and morphology with live weight, age and sex in beef-cross-dairy cattle. All animals were weighed the day before slaughter. The metacarpus and humerus were collected at slaughter and scanned at the mid-diaphysis using pQCT. Live weight was the primary explanatory variable for bone size and strength in all cohorts. However, the effect of age was significant, such that magnitude of response to liveweight was less in the 24-month-old cohort. Sex was significant within cohorts in that bulls had a shorter metacarpus than steers and heifers had a shorter metacarpus than steers at age of slaughter.

A Holistic Approach to Bone Tumors in Dogs and Cats: Radiographic and Histologic Correlation.

The skeletal system is a common site for neoplasia in dogs and cats, and primary bone tumors may develop from any of the mesenchymal tissues present in bone. Imaging and histopathology are routinely used in the diagnosis of bone tumors, and the 2 techniques are highly complementary. While imaging may be highly suggestive of a specific diagnosis and treatment may be instituted based on this, definitive diagnosis requires histopathology of either incisional or excisional biopsies or an amputation specimen. However, there are a number of diagnostic dilemmas when the pathologist interprets bone biopsy samples, such as distinguishing reactive bone and tumor bone, fracture callus and tumor bone, different benign fibro-osseous lesions, and different types of bone sarcoma. This review outlines the characteristic radiographic and histologic changes associated with these diagnostic problems to aid in resolving them. When a holistic approach is taken to evaluation of the signalment, history, and clinical, radiologic, and microscopic features, a diagnosis may be possible. The pathologist is greatly assisted in the interpretation of bone samples by having access to imaging and should routinely request either the images or the imaging reports if they are not received from submitting veterinarians.

Expression of Renal Vitamin D and Phosphatonin-Related Genes in a Sheep Model of Osteoporosis.

Osteoporosis is a significant public health issue around the world, with post-menopausal osteoporosis due to estrogen deficiency resulting in approximately ¾ of cases. In this study, 18 aged Merino ewes were ovariectomized, and 10 were controls. Three of the ovariectomized ewes were treated weekly with 400 mg of methylprednisolone for 5 months and three were treated weekly for 2 months, followed by a 3-month recovery period. At 2 months, five control animals and six ovariectomized animals were euthanized. At 5 months, all the remaining ewes were euthanized. Kidney samples were collected postmortem for qPCR analysis of NPT1, PTH1R, NPT2a, NPT2c, Klotho, FGFR1IIIc, VDR, CYP24A1, CYP27B1, TRPV5, TRPV6, CalD9k, CalD28k, PMCA and NCX1. Ovariectomized sheep had significantly greater VDR expression compared with other groups. Ovariectomized sheep treated with glucocorticoids for 2 months followed by euthanasia at 5 months showed significant differences in TRPV5, CYP24A1 and klotho gene expression compared to other groups. Differences in klotho expression were most marked after adjustment for repeated measures (p = 0.1). Klotho is known as the "anti-aging" hormone and is involved in calcium and phosphorus metabolism. Klotho may be involved in the recovery of bone mineral density in ovariectomized sheep treated with glucocorticoids for 2 months followed by euthanasia at 5 months. Further research on the role of klotho is recommended.

Expression of Phosphatonin-Related Genes in Sheep, Dog and Horse Kidneys Using Quantitative Reverse Transcriptase PCR.

The aim of this preliminary study was to determine the relative expression of phosphatonin pathway-related genes in normal dog, sheep and horse kidneys and to explore the relationships between the different genes. Kidneys were collected post-mortem from 10 sheep, 10 horses and 8 dogs. RNA was extracted, followed by reverse transcriptase quantitative polymerase chain reaction for fibroblast growth factor receptor 1 IIIc (FGFR1IIIC), sodium-phosphate co-transporter (NPT) 1 (SLC17A1), NPT2a (SLC34A1), NPT2c (SLC34A3), parathyroid hormone 1 receptor (PTH1R), klotho (KL), vitamin D receptor (VDR), 1a-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1). NPT2a was highly expressed in the dog kidneys, compared with those of the horses and sheep. NPT1 had greatest expression in horses and sheep, although the three different NPTs all had relatively similar expression in sheep. There was little variability in FGFR1IIIc expression, particularly in the dogs and horses. FGFR1IIIc expression was negatively correlated with NPT genes (except NPT2a in sheep), while NPT genes were all positively correlated with each other. Unexpectedly, klotho was positively correlated with NPT genes in all three species. These results provide the basis for further research into this important regulatory system. In particular, species differences in phosphatonin gene expression should be considered when considering the pathogenesis of chronic kidney disease.