Toward the most ideal case-control design with related and unrelated dogs in whole-exome sequencing studies.

With the recent development of whole-exome sequencing enrichment designs for the dog, a novel tool for disease-association studies became available. The aim of disease-association studies is to identify one or a very limited number of putative causal variants or genes from the large pool of genetic variation. To maximize the efficiency of these studies and to provide some directions of what to expect, we evaluated the effect on variant reduction for various combinations of cases and controls for both dominant and recessive types of inheritance assuming variable degrees of penetrance and detectance. In this study, variant data of 14 dogs (13 Labrador Retrievers and one Dogue de Bordeaux), obtained by whole-exome sequencing, were analyzed. In the filtering process, we found that unrelated dogs from the same breed share up to 70% of their variants, which is likely a consequence of the breeding history of the dog. For the designs tested with unrelated dogs, combining two cases and two controls gave the best result. These results were improved further by adding closely related dogs. Reduced penetrance and/or detectance has a drastic effect on the efficiency and is likely to have a profound effect on the sample size needed to elucidate the causal variant. Overall, we demonstrated that sequencing a small number of dogs results in a marked reduction of variants that are likely sufficient to pinpoint causal variants or genes.

Erosion of the medial compartment of the canine elbow: occurrence, diagnosis and currently available treatment options.

Erosion of the medial compartment of the elbow joint refers to full thickness cartilage loss with exposure of the subchondral bone (modified Outerbridge grades 4-5) of the medial part of the humeral condyle (MHC) and the corresponding ulnar contact area. This finding may appear in the absence of an osteochondral fragment or a cartilage flap, or in combination with fragmentation of the medial coronoid process (MCP) or osteochondritis dissecans (OCD) of the MHC. With regard to the prognosis, it is important to diagnose these severe erosions. Imaging of cartilage lesions by means of radiography, ultrasonography, computed tomography or magnetic resonance imaging is challenging in dogs. In contrast, direct arthroscopic inspection provides detailed information about the cartilage. The treatment of these severe erosions is difficult because of the limited regenerative capacity of cartilage and presumed mechanical or physical triggering factors. Several conservative and surgical treatment methods have been proposed to treat elbows with severe cartilage defects. However, due to irreversible loss of cartilage, the prognosis in these cases remains guarded.

The ligaments of the canine hip joint revisited.

Numerous conventional anatomical textbooks describe the canine hip joint, but many contradictions, in particular regarding the ligament of the femoral head, are present. This paper presents a brief overview of the different literature descriptions. These are compared with own observations that have resulted in a revised description of the anatomy of the ligament of the femoral head in the dog. To this purpose, the hip joints of 41 dogs, euthanized for reasons not related to this study and devoid of lesions related to hip joint pathology, were examined. It was observed that the ligament of the femoral head is not a single structure that attaches only to the acetabular fossa, as generally accepted, but it also connects to the transverse acetabular ligament and is complemented by a strong accessory ligament that courses in caudal direction to attach in the elongation of the acetabular notch that extends on the cranioventral surface of the body of the ischium. The description of this accessory ligament in conventional anatomical handbooks is incomplete. This description of the accessory ligament of the femoral head could support the research unravelling the etiopathogenesis of hip instability.

Combined prevalence of inherited skeletal disorders in dog breeds in Belgium.

Canine hip dysplasia (CHD), canine elbow dysplasia (CED), and humeral head osteochondrosis (HHOC) are inherited traits with uneven incidence in dog breeds. Knowledge of the combined prevalence of these three disorders is necessary to estimate the effect of the currently applied breeding strategies, in order to improve the genetic health of the population. Official screening results of the Belgian National Committee for Inherited Skeletal Disorders (NCSID) revealed that an average of 31.8% (CHD, CED, or both; n = 1273 dogs) and 47.2% (CHD, CED, HHOC, or a combination of these three diseases; n = 250 dogs) of dogs are mildly to severely affected by at least one skeletal disorder. According to the current breeding recommendations in some dog breeds in Belgium, these animals should be restricted (mild signs) or excluded (moderate to severe signs) from breeding. The introduction of genetic parameters, such as estimated breeding values, might create a better approach to gradually reduce the incidence of these complex inherited joint disorders, without compromising genetic population health.

The effects of positioning, reason for screening and the referring veterinarian on prevalence estimates of canine hip dysplasia.

Although the prevalence of canine hip dysplasia (HD) has been the subject of a number of published studies, estimates vary widely. This study evaluated several possible causes for these differences. Sixty Belgian, Dutch and German veterinarians were asked to submit all hip radiographs obtained for screening purposes (irrespective of HD status) over a 2-year period, resulting in a database of 583 dogs. Each set of radiographs was accompanied by information on the reason for screening (breeding soundness examination, clinical complaint, assistance dogs, or other reasons), and dog breed, date of birth and age. Dog positioning exerted an effect at multiple levels. The agreement among different observers regarding correct or incorrect positioning was limited and incorrect positioning itself reduced the inter-observer agreement for radiographic hip conformation. Dysplastic dogs were more commonly positioned incorrectly than non-dysplastic dogs. The clinical complaint population had a high prevalence of dysplastic dogs (>70%) compared with the breeding population (11%) and the assistance dogs (6%). There was a significantly lower prevalence of HD among cases referred by veterinarians who frequently submitted hip-extended radiographs for evaluation (P = 0.002) compared to those who refer less frequently. However, this was likely to be selection bias, as radiographs that were from dogs suspected to be dysplastic were not submitted by frequent senders. The prevalence of dysplastic dogs varied widely between breeds (16.7-71.4%). Dogs diagnosed with dysplasia were significantly older than dogs considered healthy (P = 0.001) and dogs classified as borderline dysplastic (P = 0.035). Inter-observer agreement for hip conformation was moderately low, resulting in >7% variation in prevalence estimates for dysplasia.

Comparing Norberg angle, linear femoral overlap and surface femoral overlap in radiographic assessment of the canine hip joint.

OBJECTIVES: The aim of this study was to compare Norberg angle, linear femoral overlap and surface femoral overlap in radiographs of hips accepted for official hip dysplasia screening. METHODS: Radiographs of 100 dogs (200 hips) were reviewed. The three measurements were performed in each hip according to previously described methods and thereafter correlated. RESULTS: Intra- and inter-observer reliability was very high (all >85%). The linear correlation between Norberg angle and linear femoral overlap or surface femoral overlap was 0·40 and 0·41, respectively. The linear correlation between linear femoral overlap and surface femoral overlap was 0·84. CLINICAL SIGNIFICANCE: Norberg angle cannot be compared with surface femoral overlap or linear femoral overlap, and a mathematical formula could not be computed for the comparison.

Anaesthetic arthrography of the shoulder joint in dogs.

OBJECTIVE: To evaluate the use of intra-articular anaesthesia combined with positive contrast arthrography (collectively called 'anaesthetic arthrography' [AA]) in the shoulder in order to identify and confirm the source of pain in lame dogs. METHODS: Anaesthetic arthrography was performed in 30 dogs with shoulder joint lameness with a mixture of contrast medium (iohexol: 240 mg I/ml) and a local anaesthetic (mepivacaine 2%: 20 mg/ml). The effect of intra-articular anaesthesia was evaluated by an objectified visual scoring system and the arthrograms were evaluated for their diagnostic value. RESULTS: This study showed that AA was positive in 28 out of 30 dogs. Dilution of the contrast medium with a local anaesthetic produced an arthrogram of good quality for each shoulder joint. In 16 out of 18 cases of osteochondritis dissecans, a cartilage flap could be identified following arthrography. In all cases the flap was not identified from plain radiographs. Biceps brachii tendon pathology was diagnosed on arthrography in seven dogs but was missed in two dogs. Calcification at the caudal rim of the glenoid cavity was diagnosed in three dogs, but was of clinical importance in only two dogs. In addition one infection was diagnosed during synovial aspiration. CONCLUSION: Anaesthetic arthrography of the shoulder is a simple, safe, and reliable diagnostic test to confirm shoulder joint pain and simultaneously identify a lesion. This procedure may be of particular importance in cases of occult shoulder joint lameness when clinical findings and plain radiographs are inconclusive.

Diagnostic intra-articular anaesthesia of the elbow in dogs with medial coronoid disease.

OBJECTIVE: Assessment of the effect of intra-articular anaesthesia on lameness caused by medial coronoid disease. METHODS: This study included 90 dogs that were evaluated for the complaint of unilateral forelimb lameness. All dogs were suspected of having an elbow problem for which orthopaedic examination and radiographs showed inconclusive findings. Following a short sedation, mepivacaine was injected intra-articularly to determine whether lameness was caused by a suspected elbow problem. This effect was compared with the final diagnosis based on computed tomography and arthroscopy. RESULTS: Out of 90 dogs, 78 (87%) dogs had an improvement of lameness after injection of the local anaesthetic, which confirmed the elbow joint as the primary source of lameness. A positive response was seen in all types of medial coronoid lesions. A false negative result was observed in 12 dogs (13%). CLINICAL RELEVANCE: Medial coronoid disease is represented by different types of pathologic lesions including chondromalacia, fissures, fragments, and medial compartment disease. Diagnosis may be difficult because of limited clinical or radiographic signs or a combination of both. Intra-articular anaesthesia can be a helpful diagnostic tool to localize the problem by eliminating pain and reducing lameness to a great extent in all types of coronoid lesions.

Direct action through the sertoli cells is essential for androgen stimulation of spermatogenesis.

Androgens act to stimulate spermatogenesis through androgen receptors (ARs) on the Sertoli cells and peritubular myoid cells. Specific ablation of the AR in either cell type will cause a severe disruption of spermatogenesis. To determine whether androgens can stimulate spermatogenesis through direct action on the peritubular myoid cells alone or whether action on the Sertoli cells is essential, we crossed hypogonadal (hpg) mice that lack gonadotrophins and intratesticular androgen with mice lacking ARs either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with testosterone (T) or dihydrotestosterone (DHT) for 7 d and testicular morphology and cell numbers assessed. Androgen treatment did not affect Sertoli cell numbers in any animal group. Both T and DHT increased numbers of spermatogonia and spermatocytes in hpg mice, but DHT has no effect on germ cell numbers in hpg.SCARKO and hpg.ARKO mice. T increased germ cell numbers in hpg.SCARKO and hpg.ARKO mice, but this was associated with stimulation of FSH release. Results show that androgen stimulation of spermatogenesis requires direct androgen action on the Sertoli cells.

Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors.

FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.

Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression.

Evidence from several models of hormone depletion and/or replacement and from knockout animals points to a key role of androgens in the control of spermatogenesis. In testes of mice with a Sertoli cell-selective ablation of the androgen receptor (SCARKO), transcriptional profiling, using microarray technology, revealed that, already on postnatal day 10,692 genes are differentially expressed compared with testes of control mice. Further evaluation of a subset of these genes by quantitative RT-PCR suggested that differences in expression may already be evident on day 8 or earlier. As the androgen receptor in mouse Sertoli cells becomes immunologically detectable around day 5, we tried to identify the earliest responses to androgens by a new transcriptional profiling study on testes from 6-day-old SCARKO and control mice. No obvious and novel early androgen response genes, potentially acting as mediators of subsequent indirect androgen actions, could be identified. However, several genes differentially expressed on day 10 already displayed a response to androgen receptor ablation on day 6. Quantitative RT-PCR studies for 12 of these genes on 10 paired SCARKO and control testes from 4-, 6-, 8-, 10-, 20- and 50-day-old mice revealed significant differences in expression level from day 4 onwards for three genes (Eppin, PCI, Cldn11) and from day 6 onwards for one more gene (Rhox5). For at least two of these genes (Rhox5 and Eppin), there is evidence for direct regulation via the androgen receptor. For three additional genes (Gpd1, Tubb3 and Tpd52l1) significantly lower expression in the SCARKO was noted from day 8 onwards. For all the studied genes, an impressive increase in transcript levels was observed between day 4-50 and differential expression was maintained in adulthood. It is concluded that the SCARKO model indicates incipient androgen action in mouse Sertoli cells from day 4 onwards.

Prevalence of hip dysplasia, elbow dysplasia and humeral head osteochondrosis in dog breeds in Belgium.

The official screening results of the Belgian National Committee for Inherited Skeletal Disorders, an affiliate of the Belgian Kennel Club, have been used to estimate the prevalence of hip dysplasia, elbow dysplasia and humeral head osteochondrosis in the dog breeds in Belgium, and these have been compared with reported prevalence data from other countries. In some breeds, the prevalence of hip and elbow dysplasia is very high, both in Belgium and in other countries. Comparisons of the prevalence of hip dysplasia are not always feasible because different systems are used to evaluate the quality of the hips and because there is no strict consensus on what should be considered a diseased hip joint.

Spermatogenesis and sertoli cell activity in mice lacking sertoli cell receptors for follicle-stimulating hormone and androgen.

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.

A comparison of tepoxalin-buprenorphine combination and buprenorphine for postoperative analgesia in dogs: a clinical study.

The present study compares the analgesic effect of a tepoxalin-buprenorphine combination to that of buprenorphine alone in the 24 h peri-operative period in 20 dogs undergoing cranial cruciate ligament repair, which were randomly assigned to the two treatment protocols (n = 10). Additionally, possible side effects induced by tepoxalin were investigated. Analgesia was compared using a visual analogue scale (VAS) and a multifactorial pain scale (MFPS), by an anaesthetist blinded from treatment. Analysis of the overall VAS-scores showed a significant decrease over time in both treatment groups. The decrease in the two groups was not significantly different from each other. No significant differences were found between the MFPS-scores of both protocols. Potential side effects of tepoxalin were investigated by venous blood sampling before premedication and 24 h after extubation, a buccal mucosal bleeding time test and recording of vomiting, diarrhoea and adverse effects at the surgical site. Analysis of the blood parameters showed that fibrinogen levels were overall higher 24 h after surgery in both protocols, but were significantly more elevated in the tepoxalin group. No significant differences were found for the other blood parameters. Statistically, tepoxalin failed to improve analgesia induced by buprenorphine. There was no convincing evidence that the administration of tepoxalin was not associated with gastrointestinal side effects. There were no significant adverse effects on renal function and primary haemostasis.

Interobserver agreement in the diagnosis of canine hip dysplasia using the standard ventrodorsal hip-extended radiographic method.

OBJECTIVES: To determine the agreement between observers and to investigate the effect of observer experience in diagnosing canine hip dysplasia and providing final scoring of hips using the standard ventrodorsal hip-extended radiographic method. The agreement of the final scoring, with a presumed correct assessment based on the Norberg angle, is also investigated. METHODS: Thirty observers were requested to read 50 ventrodorsal hip-extended radiographs of 25 dogs according to Federation Cynologique International criteria. Groups of experienced (nine members) and inexperienced (21 members) observers were used. RESULTS: For providing the distinction between dysplastic versus non-dysplastic dogs, the average interobserver agreement was 72 per cent and was significantly higher (P<0.0001) than the score that could be expected by chance without any agreement between observers. For providing the final score (A, B, C, D or E), an average interobserver agreement of 43.6 per cent was found. In the experienced group, an agreement score of 76 per cent was found for the distinction between AB versus non-AB and an agreement score of 81 per cent was found for the distinction between C versus non-C. The agreement score was significantly higher (P<0.0001) for the experienced group than for the inexperienced group in all cases. Agreement between the presumed correct assessment based on the Norberg angle and the observer's evaluation was low (P=0.35), irrespective of whether the observers were experienced (71.8 per cent correct assessments) or inexperienced (69 per cent correct assessments). CLINICAL SIGNIFICANCE: Although interobserver agreement is low, observer experience increases agreement.

The use of an external skeletal traction device for distal fractures in the dog. A clinical case series of 11 patients.

Metacarpal and metatarsal fractures in 11 patients were treated 'closed' with a specially designed External Skeletal Fixation (ESF) frame with a walking bar and traction applied to the digits. Mediolateral angulation had improved postoperatively in 10 of the 11 patients. Craniocaudal angulation had improved in eight patients and could not be evaluated in three due to ESF frame superimposition. At follow-up, eight patients had a good clinical function, whereas three patients were still slightly lame. Only minor ESF-related complications were seen (pin loosening, pin tract infection, cerclage wire breakage and bending of the frame), which resolved without intervention after frame removal.

Swimmer syndrome in a Devon rex kitten and an English bulldog puppy.

A three-week-old Devon rex kitten and a four-week-old English bulldog puppy were presented with "swimmer syndrome". The owners consulted several veterinarians who suggested euthanasia as the only possible solution for this condition. Physiotherapy in the puppy, and physiotherapy and bandaging in the kitten led to the resolution of the clinical signs and resulted in normal ambulation after several weeks. The authors concluded that intensive physiotherapy and dedication of the owner can lead to success.

A phase II study of induction therapy with carboplatin and gemcitabine among patients with locally advanced non-small cell lung cancer.

INTRODUCTION: The objectives of this trial were to evaluate the activity and safety of gemcitabine carboplatin as induction therapy in patients with locally advanced non-small cell lung cancer METHODS: Patients received two cycles of gemcitabine (1250 mg/m on day 1 and 8), plus carboplatin (area under the curve = 5 on day 1), after which response was established. Patients received a third course only in the case of an objective response (OR). Non-responding patients were directly irradiated. Toxicity was assessed according to the NCI-CTC version 2, radiation toxicity was assessed according to RTOG criteria. Response evaluation was performed according to RECIST criteria. RESULTS: We identified 42 patients, of whom 37 were eligible. Of these, 51% (95% CI, 34%-68%) achieved an OR, all partial responses. No disease progression on therapy was established. Toxicity was mostly hematological: 35% trombocytopenia grade 3 and 4, and 40% neutropenia grade 3 and 4. No severe bleeding or hospitalization because of febrile neutropenia occurred. CONCLUSIONS: Gemcitabine and carboplatin administered according to a 3-week schedule is an active and safe induction regimen. Pending the results of a phase III study, we believe that it is a reasonable alternative among patients for whom cisplatin-based chemotherapy is contraindicated.

Effects of atorvastatin and vitamin E on lipoproteins and oxidative stress in dialysis patients: a randomised-controlled trial.

OBJECTIVES: The objective of this study was to examine the effects of treatment with atorvastatin, alpha-tocopherol and the combination of both, on lipoproteins and oxidative stress in dialysis patients. DESIGN AND SETTING: This double-blind randomised placebo-controlled trial was performed at the dialysis department of a non-university hospital. SUBJECTS, INTERVENTION AND MEASUREMENTS: A total of 44 clinically stable, non-diabetic patients on dialysis therapy (23 on haemo- and 21 on peritoneal-dialysis) without manifest cardiovascular disease were included in this study. They were randomised for treatment during a period of 12 weeks with 40 mg atorvastatin + placebo alpha-tocopherol (group 1) once daily, 800 IU alpha-tocopherol + placebo atorvastatin once daily (group 2), 40 mg atorvastatin + 800 IU alpha-tocopherol once daily (group 3), or placebo atorvastatin + placebo alpha-tocopherol once daily (group 4). Assessment of lipid profile and oxidative stress was performed at the start of the study and after 12 weeks of treatment. RESULTS: Treatment with atorvastatin reduced total cholesterol, triglycerides (TG), low-density lipoprotein (LDL) cholesterol, apolipoprotein B (apoB) and levels of oxidised LDL (oxLDL) with 30-43%. It had no influence on LDL oxidisability. Additional supplementation with alpha-tocopherol had no effect on lipid profile and oxLDL levels but decreased in vitro LDL oxidisability. No side-effects were observed. CONCLUSIONS: Treatment with atorvastatin is effective in lowering plasma total cholesterol, TG, LDL, apoB and oxLDL in a population of stable dialysis patients and might therefore be an effective tool in improving the poor cardiovascular outcome in these patients. Supplementation of alpha-tocopherol to atorvastatin had beneficial effects on in vitro LDL oxidisability and might therefore be of additional value. Further research on the clinical effects of treatment with atorvastatin in combination with alpha-tocopherol is necessary.