A preliminary investigation of the subcutaneous tissue reaction to a 3D printed polydioxanone device in horses.

BACKGROUND: A 3D printed self-locking device made of polydioxanone (PDO) was developed to facilitate a standardized ligation technique. The subcutaneous tissue reaction to the device was evaluated after implantation in ten horses of mixed age, sex and breed and compared to loops of poly(lactic-co-glycolic acid) (PLGA). In two of the horses, the implants were removed before closing the skin. The appearance of the implants and surrounding tissue was followed over time using ultrasonography. Implants were removed after 10 and 27 (± 1) days for histologic examination. RESULTS: On macroscopic inspection at day 10, the PDO-device was fragmented and the surrounding tissue was oedematous. On ultrasonographic examination, the device was seen as a hyperechoic structure with strong acoustic shadowing that could be detected 4 months post-implantation, but not at 7 months. Histology revealed a transient granulomatous inflammation, i.e., a foreign body reaction, which surrounded both PDO and PLGA implants. The type and intensity of the inflammation varied between individuals and tissue category. CONCLUSIONS: The 3D printed PDO-device caused a transient inflammatory reaction in the subcutaneous tissue and complete resorption occurred between 4 and 7 months. Considering the intended use as a ligation device the early fragmentation warrants further adjustments of both material and the 3D printing process before the device can be used in a clinical setting.

In Vivo Versus In Vitro Degradation of a 3D Printed Resorbable Device for Ligation of Vascular Tissue in Horses.

A resorbable 3D printed polydioxanone (PDO) device is manufactured to facilitate ligation of vascular tissue during surgery. The device must provide sufficient mechanical performance throughout the healing period. Therefore, degradation and mechanical performance of the device are investigated as a function of in vivo and in vitro aging. During aging the PDO device released cyclic and linear water-soluble products. In vivo aging resulted in higher relative number of linear oligomers in comparison to in vitro aging. A major loss of mechanical performance is observed after only 10 days in vivo and the Young's modulus (E) and tensile strength at break (σb ) decreased by 28% and 54%, respectively. This is in contrast to in vitro aging, where no loss of mechanical properties is observed during the same period. The in vivo aged devices exhibit clear holes in the matrices after 28 days, while apparent cracks are observed first after 140 days in vitro. These results highlight the sensitivity of the degradation process of resorbable devices with regards to the interactions of the device with the surrounding environment (tissues) and demonstrate the importance of in vivo testing as compliment to in vitro testing before clinical use of devices.

Equine saliva components during mastication, and in vivo pH changes in the oral biofilm of sound and carious tooth surfaces after sucrose exposure.

BACKGROUND: The role of saliva composition and dietary sugar in development of infundibular caries in equine cheek teeth is not fully understood. This study analysed electrolyte and urea concentrations in saliva in relation to different forage and measured pH changes after sucrose application in vivo in sound and carious cheek teeth. RESULTS: Forage type had no effect on the equine saliva electrolyte concentrations, which varied considerably both intra- and inter-individually. Chewing resulted in increased values for all electrolytes except bicarbonate. Compared with stimulated human saliva, horse saliva after mastication, contained higher amounts of potassium, calcium and bicarbonate, and less phosphate. The in vivo pH measurements showed a lower resting pH and a more pronounced pH drop after sucrose application in carious teeth compared to sound teeth. CONCLUSIONS: No large differences were found between the composition of equine saliva and human saliva. A more pronounced acidogenicity was found for the carious than sound teeth. Thus, the caries process in equine cheek teeth seems to follow the same pattern as in human teeth, caused by acid production by oral microorganisms after sugar consumption.

Secretory immunoglobulin A and immunoglobulin G in horse saliva.

This study aimed to increase the knowledge on salivary antibodies in the horse since these constitute an important part of the immune defence of the oral cavity. For that purpose assays to detect horse immunoglobulin A (IgA) including secretory IgA (SIgA) were set up and the molecular weights of different components of the horse IgA system were estimated. Moreover, samples from 51 clinically healthy horses were tested for total SIgA and IgG amounts in saliva and relative IgG3/5 (IgG(T)) and IgG4/7 (IgGb) content were tested in serum and saliva. Results showed a mean concentration of 74µg SIgA/ml horse saliva and that there was a large inter-individual variation in salivary SIgA concentration. For total IgG the mean concentration was approx. 5 times lower than that of SIgA, i.e. 20µg IgG/ml saliva and the inter-individual variation was lower than that observed for SIgA. The saliva-serum ratio for IgG isotypes IgG3/5 and IgG4/7 was also assessed in the sampled horses and this analysis showed that the saliva-serum ratio of IgG4/7 was in general approximately 4 times higher than that of IgG3/5. The large inter-individual variation in salivary SIgA levels observed for the normal healthy horses in the present study emphasises the need for a large number of observations when studying this parameter especially in a clinical setting. Moreover, our results also indicated that some of the salivary IgG does not originate from serum but may be produced locally. Thus, these results provide novel insight, and a base for further research, into salivary antibody responses of horses.

Large Deletions at the SHOX Locus in the Pseudoautosomal Region Are Associated with Skeletal Atavism in Shetland Ponies.

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160-180 kb and 60-80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development.

Ossification of ungular cartilages in front feet of cold-blooded trotters--a clinical radiographic evaluation of development over time.

BACKGROUND: It has not yet been shown that ossification of ungular cartilages (OUC) is a pathological condition. Beside heredity, factors such as sex, age, repeated concussion, local trauma, hoof and body size have been suggested as contributing factors for OUC development. By comparing radiographs of front hooves from cold-blooded trotters with different age we wanted to evaluate when development of OUC in cold-blooded trotters occurs and if and when it stabilizes in relation to age and workload. Diagnosis and grading of OUC were based on radiological field examinations of 649 Swedish and Norwegian cold-blooded trotters' front hooves. A hundred and forty-seven of the horses were re-examined 3-13 years (mean age 9, median 8 years) after the first occasion. All radiographs were evaluated blind, using two different grading systems for OUC. Work load, in form of number of races completed, and body size score were collected from official data. Four statistical ordinal regression models were used, compared and evaluated. RESULTS: We identified a breakpoint at 2.8 ± 0.38 years of age when ossification ends and proposed a simpler grading system with more consistent results. There was no significant correlation between body size and grade of OUC. Comparison of different statistical methods for evaluation of ordinal data revealed a piecewise linear regression model as most suitable. CONCLUSIONS: Individuals with OUC developed this condition during the stage of life when their hooves develop in size. Results from this study can assist equine practitioners when examining and for understanding this condition in their clinical work and is also beneficial for the Scandinavian equine industry when devising breeding programs.

Significance of ossificated ungular cartilages regarding the performance of cold-blooded trotters.

BACKGROUND: Ossification of the ungular cartilages (OUC) in the foot of horses has been studied for more than 100 years. There is a high heritability of this condition but its clinical relevance has remained questionable. Nevertheless, modern equine orthopedic literature ranks OUC as one of top 10 causes of lameness in cold-blooded trotters and stallions of these breeds are excluded from breeding if they have more than mild levels of side bones. Cold-blooded trotters have been used for racing for many decades and official sports data have been available since 1923. A decreased performance is often the only obvious clinical sign noticed by trainers and owners motivating them to seek professional help from veterinarians and farriers. By comparing various performance parameters in Swedish-Norwegian cold-blooded trotters without and with different grades of OUC, we aimed to determine the clinical relevance of ossified hoof cartilages in a population of high-performance horses. Front hooves from 649 Swedish-Norwegian cold-blooded trotters were evaluated radiologically regarding OUC. Breeding index and official sports data originating from strict protocols kept by groups of officials in trotting associations was used for comparison of performance of these horses that together had competed in more than 23,000 races between 1973 and 2009. Generalized linear mixed models were used for the statistical analyses. The response variable was modeled using ordinal logistic models with a multinomial distribution and a cumulative logit link function. The horse was used as a random factor. RESULTS: Significant effects of gender on performance were demonstrated, but no correlations were found between different positions nor grades of ossified ungular cartilage and number of starts, running pace, race winnings, number of races completed in a regular gait. CONCLUSIONS: Ossification of the ungular cartilages does not cause decreased performance in cold-blooded trotters and is therefore most likely not a cause of clinical or subclinical lameness in this breed. Results from this study can assist equine professionals in evaluating and interpreting the clinical relevance of radiological findings on ossified hoof cartilage among heavy and high-performing horses.

The prevalence of oral ulceration in Swedish horses when ridden with bit and bridle and when unridden.

Oral soft tissue ulcers are common disorders of horses, but it is unclear if their prevalence is increased by riding horses with a bit and bridle. Oral examinations were performed on 113 horses and ponies, all which had received routine dental floating, that were divided into four groups depending on when they had last been ridden with a bit and bridle. The subjects comprised: group 1, a randomly selected population of ridden horses; group 2, a group of horses examined after being rested at pasture for 5 weeks; group 3, the previous group following 7 weeks of riding with a bit and bridle, and group 4, brood mares that had not been ridden for at least 11 months. Lip and intraoral soft tissue lesions were recorded at seven pre-determined locations, with lesions classified as large or small; acute or chronic. The examinations showed that horses that were currently being ridden with a bit and bridle had a significantly higher prevalence of large and acute buccal ulcers opposite the maxillary Triadan 06 teeth and of the commissures of the lips, as compared to horses that were not being currently ridden. It was concluded that using a bit and bridle can cause oral ulceration even in horses that have regular prophylactic dental floating. It is suggested that riding tack should be individually fitted for each horse and also that prophylactic dental treatments should be individually adapted for each horse.

Veterinary-care events and costs over a 5-year follow-up period for warmblooded riding horses with or without previously recorded locomotor problems in Sweden.

Our objective was to determine the amount, type and cost of veterinary care during a 5-year follow-up period, in Swedish warmblooded riding horses with and without locomotor problems as recorded in 1997. The study population comprised horses with complete insurance both for veterinary care and life during 1997. The horses were followed from the beginning of January 1998 to the end of December 2002. The exposed cohort was composed of 1558 horses that had experienced >or=1 veterinary-care event because of locomotor problems in 1997 (LP-cohort). The exposure-negative (LN) cohort was composed of 16,513 horses that had no recorded locomotor problem during 1997. For the outcome of locomotor problems during the follow-up period, stratified incidence proportions and a repeated-measures multivariable-logistic regression model in general produced the same conclusions. An interaction between year and cohort showed larger odds ratios (ORs) in the first year of follow-up compared to later (OR 4.8 and 1.2 in the LP- and LN-cohort, respectively), but a smaller difference during the subsequent years (in the 5th year OR 1.8 and 1.0, respectively). Horses with life-insurance values of >30,000 SEK in the LP- and LN-cohorts had ORs of 4.7 (99% CI 3.9, 5.6) and 2.6 (99% CI 2.3, 2.9), respectively. For life-insurance values of

Caries in the infundibulum of the second upper premolar tooth in the horse.

BACKGROUND: Swedish equine dental practices have empirically found that the prevalence of infundibular caries as a primary disorder in the first permanent premolar teeth (P2) of the horse upper jaw has increased during the last 10 years. A previously unknown bacterial species, Streptococcus devriesei (CCUG 47155T), which is related to Streptococcus mutans, has recently been isolated from these carious lesions. To understand the aetiology of caries in horses, it is essential to elucidate the relationship between S. devriesei and P2 infundibular caries. METHODS: The anterior infundibulum of maxillary P2, or the occlusal surface at the site of the infundibulum, in 117 horses and ponies, 77 with and 40 without caries in this tooth, was sampled for bacteriological analyses between 1990 and 2004. Samples were transported in VMGA III medium and then inoculated onto MSB agar. The approximate number of bacteria was counted in each sample and the isolates were characterised biochemically, using a commercial kit. RESULTS: All 50 samples taken from carious lesions after 2002 were positive for an S. mutans-like strain, i.e. S. devriesei. The bacteria were also found in four of the control animals, but were much less numerous than in samples from caries-affected horses. None of the swabs taken prior to 2002 were positive for this bacteria. CONCLUSION: Our results demonstrate that S. devriesei can colonise the infundibulum of P2 of the horse upper jaw, which can be fatal for the dental tissue. We conclude that S. devriesei is strongly associated with P2 caries in horses.

Streptococcus devriesei sp. nov., from equine teeth.

Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, alpha-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a close affinity with Streptococcus mutans, Streptococcus ferus and related organisms. Sequence divergence values of > 5% with these and other reference streptococcal species however demonstrated the organisms from equine sources represent a novel species. Based on the phenotypic distinctiveness of the new bacterium and molecular chemical and molecular genetic evidence, it is proposed that the unknown species be classified as Streptococcus devriesei sp. nov. The type strain of Streptococcus devriesei is CCUG 47155T (= CIP 107809T).