[Development of a real-time RT-PCR for detection of equine influenza virus]. / Entwicklung einer real-time RT-PCR zum Nachweis von equinem Influenzavirus.

Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages.

Maturation of the equine medial femoral condyle osteochondral unit.

Objective: The juvenile equine medial femoral condyle (MFC) is frequently affected with radiographic changes (sclerosis and subchondral lucencies) that arise at a similar site to juvenile osteochondritis dissecans (JOCD) in children. There is little information on maturation of the MFC. To describe the normal development of the equine MFC osteochondral unit from birth to 2 years. Methods: Micro CT, histology and immunohistochemistry were performed on healthy equine MFCs (n = 29) at sites where lesions occur. Parameters assessed included: cartilage thickness; the epiphyseal growth plate cartilage organization; the osteochondral junction and progression of endochondral ossification. Results: From 0 to 6 months, chondrocytes near the articular surface are small and flat and have a characteristic hypertrophic appearance near the osteochondral junction but are not arranged in columns like physeal growth plates. The osteochondral junction is also crossed by cartilage canals containing vessels giving a porous appearance on 3D µCT images. At 7 months of age, a subchondral bone plate compact structure emerged histologically coincident with the end of endochondral ossification (absence of type X collagen immunostain and chondrocyte hypertrophy). Conclusion: New information is provided on MFC osteochondral unit maturation that will improve our understanding of the development of juvenile equine orthopaedic disease. Equine MFC endochondral ossification is complete at 6 months of age. The immature osteochondral junction may be structurally fragile because of its microarchitecture and susceptible to focal traumatic events that induce developmental lesions.

Role of endochondral ossification of articular cartilage and functional adaptation of the subchondral plate in the development of fatigue microcracking of joints.

The mechanisms that regulate functional adaptation of the articular ends of long bones are poorly understood. However, endochondral ossification of articular cartilage and modeling/remodeling of the subchondral plate and epiphyseal trabeculae are important components of the adaptive response. We performed a histologic study of the distal end of the third metacarpal/metatarsal bone of Thoroughbreds after bones were bulk-stained in basic fuchsin and calcified sections were prepared. The Thoroughbred racehorse is a model of an extreme athlete which experiences particularly high cyclic strains in distal limb bones. The following variables were quantified: microcrack boundary density in calcified cartilage (N.Cr/B.Bd); blood vessel boundary density in calcified cartilage (N.Ve/B.Bd); calcified cartilage width (Cl.Cg.Wi); duplication of the tidemark; and bone volume fraction of the subchondral plate (B.Ar/T.Ar). Measurements were made in five joint regions (lateral condyle and condylar groove; sagittal ridge; medial condylar and condylar groove). N.Cr/B.Bd was site-specific and was increased in the condylar groove region; this is the joint region from which parasagittal articular fatigue (condylar) fractures are typically propagated. Formation of resorption spaces in the subchondral plate was co-localized with microcracking. N.Ve/B.Bd was also site-specific. In the sagittal ridge region, N.Ve/B.Bd was increased, Cl.Cg.Wi was decreased, and B.Ar/T.Ar was decreased, when compared with the other joint regions. Multiple tidemarks were seen in all joint regions. Cumulative athletic activity was associated with a significant decrease in B.Ar/T.Ar in the condylar groove regions. N.Cr/B.Bd was positively correlated with B.Ar/T.Ar (P < 0.05, r(s) = 0.29) and N.Ve/B.Bd was negatively correlated with B.Ar/T.Ar (P < 0.005, r2 = 0.14) and Cl.Cg.Wi (P < 0.05, r2 = 0.07). We conclude that endochondral ossification of articular cartilage and modeling/remodeling of the subchondral plate promote initiation and propagation of site-specific fatigue microcracking of the joint surface, respectively, in this model. Microcracking of articular calcified cartilage likely represents mechanical failure of the joint surface. Propagation of microcracks into the subchondral plate is a critical factor in the pathogenesis of articular condylar fatigue (stress) fracture. Functional adaptation of the joint likely protects hyaline cartilage from injury in the short-term but may promote joint degeneration and osteoarthritis with ongoing athleticism.

Carpal and fetlock conformation of the juvenile Thoroughbred from birth to yearling auction age.

REASONS FOR PERFORMING STUDY: There is little information available about conformational changes in the forelimbs of growing foals. OBJECTIVES: To describe the conformation of the carpus and fetlock of Thoroughbred foals from birth to yearling sale age. METHODS: Subjective assessments of the fetlock and carpal conformation of 119 Thoroughbred foals were made within the first month of life and then at 30 day intervals until at least age 120 days. At least 70 subjects were examined further at 60 day intervals until September of their second year. Conformation grades are reported for 5 age groups: first 7 days and at a mean of 46, 176, 362 and 525 days. The conformation of all available sires and dams of subjects was also graded. RESULTS: All subjects demonstrated carpal deviations, such as valgus, outward rotation and offset, and approximately 30% had fetlock deviations. Heavier birth weights were associated with carpal offset and fetlock inward conformation at most ages, and heavier yearlings were more likely to be carpal valgus. The carpal conformation of the sire (offset and outward rotation) was associated with similar yearling carpal conformation. During the study period, the carpal conformation of Thoroughbred foals became less valgus and more offset. Fetlock conformation became more inwardly deviated during the first 6 months of the study. CONCLUSIONS: Carpal and fetlock conformation change greatly in Thoroughbred foals up to age 18 months. The phenotype of the sire can be associated with yearling carpal conformation and bodyweight, particularly at birth and as yearlings, is associated with yearling fetlock and carpal conformation. POTENTIAL RELEVANCE: New factors associated with forelimb conformational deviations have been identified that may help breeders better to manage young racing stock.

Up-regulation of site-specific remodeling without accumulation of microcracking and loss of osteocytes.

Functional adaptation of bone normally protects the skeleton from fracture during daily activity. Accumulation of microcracking and loss of osteocytes have been implicated in the regulation and initiation of targeted (reparative) remodeling of bone and, in certain situations, the development of fatigue or stress fracture. We performed a histologic study of the dorsal cortex of the mid-diaphysis of the third metacarpal (Mc-III) bone of Thoroughbred racehorses after bones were bulk-stained in basic fuchsin and transverse calcified sections were prepared. The Thoroughbred racehorse is an extreme athlete whose Mc-III bone experiences particularly high cyclic strains during training and racing. A group of non-athletic horses was also included in the experiment. The following variables were quantified: activation frequency (Ac.f); bone formation rate (BFR); resorption space density (Rs.N/T.Ar); microcrack density (Cr.Dn); microcrack mean length (Cr.Le); microcrack surface density (Cr.S.Dn); osteocyte density (Ot.N/T.Ar; Ot.N/B.Ar); and bone volume fraction (B.Ar/T.Ar). Ac.f and BFR were estimated using a mathematical algorithm. Using confocal microscopy, bones were examined for fine microcracks, diffuse matrix injury, and disruption of the osteocyte syncytium. Low values for Cr.Dn (#/mm2) were found in both groups (0.022+/-0.008 and 0.013+/-0.006 for racing Thoroughbreds and non-athletic horses, respectively). There was no significant relationship between Cr.Dn and Ot.N/T.Ar; Ot.N/B.Ar, B.Ar/T.Ar, and Ot.N/T.Ar; Ot.N/B.Ar, and remodeling (Ac.f, Rs.N/T.Ar) and Ot.N/T.Ar; Ot.N/B.Ar. Intense remodeling of the Mc-III dorsal cortex was found in the racing Thoroughbreds (Ac.f 12.8+/-7.4 #/mm2/year; BFR 31.5+/-15.6%; Rs.N/T.Ar 0.19+/-0.09 #/mm2) and was significantly increased compared with non-athletic horses. Overall, remodeling was weakly correlated with Cr.Dn (r2=0.15, P<0.05). Subtle matrix injury, not detectable by bright-field microscopy, was particularly evident adjacent to resorption spaces in Thoroughbred bone. In non-athletic horses, disruption of the dendritic cell processes of osteocytes associated with cement lines and interstitial fragments was more evident. Taken together, these findings suggest that site-specific (targeted) induction of remodeling during functional adaptation of bone in a high-strain skeletal site is not dependent on accumulation of microcracking or loss of osteocytes. We hypothesize that athleticism can directly influence bone turnover in this extreme athlete through pathways that do not involve classical linear microcracks.

Continuous absorbable suture pattern in the closure of ventral midline abdominal incisions in horses.

A retrospective study was performed to evaluate the healing of ventral midline abdominal incisions, closed with a simple continuous suture pattern using absorbable suture material, in 139 horses and foals. Dehiscence and incisional hernia developed separately in two horses. The low incidence of dehiscence and incisional hernia, compared with their reported incidence following the use of interrupted suture repair, leads the authors to recommend this alternative method of abdominal incision closure in horses. The security of closure is not sacrificed and the advantages of a rapid closure are desirable.

Incidence of the endothelin receptor B mutation that causes lethal white foal syndrome in white-patterned horses.

OBJECTIVE: To determine incidence of the Ile118Lys endothelin receptor B (EDNRB) mutation responsible for overo lethal white syndrome (OLWS) and its association with specific types of white patterning. ANIMALS: 945 horses of white-patterned bloodlines and 55 solid-colored horses of other breeds. PROCEDURE: Horses were genotyped by use of allele-specific polymerase chain reaction to determine incidence of the Ile118Lys EDNRB mutation. RESULTS: Genotypes detected were homozygous Ile118, homozygous Lys118, and heterozygous. All foals with OLWS were homozygous for the Ile118Lys EDNRB mutation, and adults that were homozygous were not found. White patterning was strongly associated with EDNRB genotype. Color patterns with highest incidence (> 94%) of heterozygotes were frame overo, highly white calico overo, and frame blend overo. White-patterned bloodlines with lowest incidence of heterozygotes (< 21 %) were tobiano, sabino, minimally white calico overo, splashed white overo, nonframe blend overo, and breeding-stock solid. The mutation was not detected in solid-colored horses from breeds without white patterning. CONCLUSIONS AND CLINICAL RELEVANCE: In homozygotes, the Ile118Lys EDNRB mutation causes OLWS. In heterozygotes, the mutation is usually responsible for a frame overo phenotype. The frame pattern can be combined with other white patterns, making accurate estimation of EDNRB genotype by visual inspection difficult. Wide range of incidence of heterozygotes in various subtypes of white-patterned horses indicates different genetic control of these color patterns. Determination of EDNRB genotype by use of a DNA-based test is the only way to determine with certainty whether white-patterned horses can produce a foal affected with OLWS.

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors.

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.

Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses.

OBJECTIVE: To determine the likely mode of inheritance and identify probable foundation horses for recurrent exertional rhabdomyolysis (RER) in Thoroughbred (TB) racehorses. ANIMALS: 4 families of TB racehorses with a high prevalence of RER, consisting of 3 to 53 horses/family, were used to determine mode of inheritance. Sixty-two TB horses with RER and 34 control TB racehorses without RER were used to identify probable foundation horses for the RER trait. PROCEDURE: RER was diagnosed by a veterinarian and verified by detecting high serum creatine kinase activity. Pedigrees dating from 1930 for all horses were entered into a database. Pedigrees of horses in 4 families were visually inspected for a pattern of inheritance and used for calculation of foundation horse contributions and inbreeding coefficients. The Markov chain Monte Carlo technique was used to analyze pedigrees of the 62 affected and 34 control horses for the conditional probability of foundation genotypes. A dominant mode of inheritance with variable expression model was used. RESULTS: Pedigree analysis supported an autosomal dominant mode of inheritance with variable expression. All affected horses from the 4 families shared a common ancestor. This ancestor and 5 other stallions had a conditional probability of 1.00 for being affected. All 6 stallions shared a common male ancestor within 3 to 5 generations. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of this study, the RER trait has been in TB racehorses for more than 70 years and may be inherited as an autosomal dominant trait with variable expression.

Successful pregnancy after partial hysterectomy in two mares.

Infertility was associated with a uterine mass in 2 Thoroughbred mares. Both mares had produced live foals, but had been barren for 2 years, despite repeated breedings. Each mass was removed through a right ventral paramedian incision. Histologic examination of the masses revealed a leiomyoma and an ovarian abscess. In 1 mare, approximately 30% of 1 uterine horn was removed because of tumor attachment; in the other mare, approximately 50% of 1 uterine horn was removed because of adhesions to the ovarian abscess. Pregnancy information was available for both mares for the 3 years after surgery; each mare had produced 2 healthy foals and was pregnant with a third.