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.

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.

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.

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.

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.

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.

The hypoattenuating ocular lens on CT is not always due to cataract formation.

Hypoattenuating ocular lenses on CT have been described with cataract formation in humans, however published studies are currently lacking regarding this finding in veterinary patients. The purpose of this retrospective and prospective study was to describe the varying CT appearances of the ocular lens in vivo, and investigate the causes for CT density variations in a population of cats and dogs. A total of 102 canine and feline patients with CT of the head acquired at the authors' hospital between May 2011 and March 2019 were included. A bilateral hypoattenuating halo surrounding an isoattenuating to mildly hypoattenuating core was described in the ocular lens center of every cat in which a Philips brand proprietary image construction filter was used. A similar but more varied hypoattenuating region was noted in the lenses of 45.8% of dogs where the same filter was applied, as well as 43.8% of dogs with a second, similar filter. Ophthalmic examination of three live cats and one dog with hypoattenuating lenses demonstrated normal lens translucency, excluding the presence of cataract. The effect of different proprietary filters on lens appearance was also described in three fresh cadavers with normal lenses identified on ophthalmic, macroscopic, and microscopic examination. Etiology of the hypoattenuating areas within the ocular lens was not conclusively determined. Recognition that such a variant may be seen in the absence of cataract is important, in order to prevent misdiagnosis.

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression.

BACKGROUND: As the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors. METHODS: The bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs. FINDINGS: There were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18. CONCLUSIONS: Universal blood donor- type Standardbred MSCs from passages 2-4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Does Juvenile Play Programme the Equine Musculoskeletal System?

In mammals, play behaviour appears innate and, because of this, may provide insight into the frequency and intensity of load that is required to stimulate positive musculoskeletal development. The objective of this review was to explore the interaction between play and tissue (bone) development at a molecular through to whole-animal level, with specific focus on the horse as a model. The basis of our understanding of the response of bone to loading is the mechanostat theorem. This assumes that at a tissue level, bone attempts to keep localised strain within the physiological range of 1500-2500 microstrain. Loads above this range result in a modelling response to reduce strain, and strain below this threshold results in remodelling to maintain the localised physiological range. In foals, locomotor play is dramatic and vigorous, with cumulative increases in both intensity and complexity. Based on published literature describing locomotor play in foals and the microstrain at different gaits in the horse, it was proposed that locomotor play in foal aligns with the mechanostat theorem in both the magnitude and frequency of load cycles applied. The cumulative increases in the complexity and intensity of locomotor play as the foal develops, in turn, ensure the strain rates associated with play remain above the local physiological range and promote material and architectural changes in the distal limb bones. Thus, spontaneous locomotor play may be vital to ensure optimal bone development in the horse. Modern management systems need to provide appropriate opportunities for foals to perform spontaneous locomotor play to optimise bone development and reduce the risk of future musculoskeletal injury later in life.

ß-Mannosidosis in German Shepherd Dogs.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid-Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was ß-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.

Draft Whole-Genome Sequences of Five Klebsiella pneumoniae Isolates from the Subantarctic Islands of New Zealand.

Klebsiella pneumoniae is a Gram-negative bacterium that can be found in the environment, as well as on mucosal surfaces of humans and animals. Here, we report the genome sequence of five K. pneumoniae isolates from substrate samples and bird feces collected in the Subantarctic Islands of New Zealand.

Draft Whole-Genome Sequences of Seven Isolates of Klebsiella pneumoniae from New Zealand Sea Lions.

Klebsiella pneumoniae is a Gram-negative bacterium that may cause infection in a broad range of hosts. We report here the genome sequences of seven K. pneumoniae isolates from New Zealand sea lions.

Interspecies differences in plasma concentrations of 25-hydroxyvitamin D3 and dermal Vitamin D synthesis of kiwi (Apteryx mantelli), tuatara (Sphenodon punctatus), and New Zealand sea lions (Phocarctos hookeri).

Vitamin D plays a central role in calcium homeostasis of most vertebrates, and is obtained in different species through diet, dermal synthesis, or a combination of both. The aim of this study was to determine the predominant routes of Vitamin D synthesis in three disparate species, brown kiwi (Apteryx mantelli), tuatara (Sphenodon punctatus), and New Zealand sea lions (Phocarctos hookeri). We surveyed plasma concentrations of 25-hydroxyvitamin D2 and D3, analysed environmental conditions and life history factors, and determined the ability of skin samples to synthesise Vitamin D3 on exposure to ultraviolet-B radiation. There was variation in the plasma/serum 25-hydroxyvitamin D3 concentrations between and within the species studied, with wild kiwi having the lowest concentrations and NZ sea lions the highest. Kiwi skin produced small but measurable amounts of Vitamin D3, while tuatara skin produced Vitamin D3 concentrations higher than that of kiwi. New Zealand sea lion skin produced the highest amount of Vitamin D3 and differed from the other two species in this study in that Vitamin D3 was present in skin before UV-B exposure. The results from this study show that all three species studied retained the ability to use both dietary and dermal sources of Vitamin D, although there was interspecies variation in the magnitude of dermal synthesis. Comparisons between these species show that there are differences in their Vitamin D pathways, but suggest that there are more factors contributing to these pathways than might be expected solely from life history characteristics.