Low microbial biomass within the reproductive tract of mid-lactation dairy cows: A study approach.

The microbiome from the reproductive tract is being investigated for its putative effect on fertility, embryo development, and health status of the human or animal host postpartum. Besides the presence of a vaginal microbiome, recent studies have claimed the existence and putative role of the uterine microbiome. Yet, the extremely low bacterial numbers and high eukaryotic/prokaryotic DNA ratio make this a highly challenging environment to study with next-generation sequencing (NGS) techniques. Here, we describe the methodological challenges that are typically encountered when performing an accurate analysis of low microbial biomass samples, illustrated by data of our own observational study. In terms of the research question, we compared the microbial composition throughout different parts of the reproductive tract of clinically healthy, mid-lactation Holstein-Friesian cows. Samples were collected from 5 dairy cows immediately after killing. Swabs were taken from the vagina, and from 4 pre-established locations of the uterine endometrium. In addition to the conventional DNA extraction blank controls, sterile swabs rubbed over disinfected disposable gloves and the disinfected surface of the uterus (tunica serosa) before incision were taken as sampling controls. The DNA extraction, DNA quantification, quantitative PCR of the 16S rRNA genes, and 16S rRNA gene sequencing were performed. In terms of NGS data analysis, we performed prevalence-based filtering of putative contaminant operational taxonomic units (OTU) using the decontam R package. Although the bacterial composition differed between the vagina and uterus, no differences in bacterial community structure (α and ß diversity) were found among the different locations in the uterus. At phylum level, uterine samples had a greater relative abundance of Proteobacteria, and a lesser relative abundance of Firmicutes than vaginal samples. The number of shared OTU between vagina and uterus was limited, suggesting the existence of bacterial transmission routes other than the transcervical one to the uterus. The mid-lactation bovine genital tract is a low microbial biomass environment, which makes it difficult to distinguish between its constitutive versus contaminant microbiome. The integration of key controls is therefore strictly necessary to decrease the effect of accidentally introduced contaminant sequences and improve the reliability of results in samples with low microbial biomass.

Dietary supplementation of micro-encapsulated sodium butyrate in healthy horses: effect on gut histology and immunohistochemistry parameters.

BACKGROUND: As colic and intestinal disorders are a major concern in horses, the aim of the present study was to investigate the effect of dietary supplementation of butyrate, known to have a diverse array of beneficial effects on intestinal health. The effect of micro-encapsulated sodium butyrate supplementation on gut histology and immunohistochemistry parameters was studied in 14 healthy warmblood horses destined for slaughter in two separate periods. Horses were fed a low fiber - high starch diet, designed to induce subsequent starch overflow in the large intestine, aiming to create a mild challenge for large intestinal health. Treatment included supplementation with either micro-encapsulated sodium butyrate (Excential Butycoat®, Orffa, Werkendam, the Netherlands) or placebo (containing only coating material). The horses were fed for 20 consecutive days at a dosage of 0.4 g/kg BW (body weight). At day 21, the horses were slaughtered and intestinal samples were collected for determination of gut pH, villus length, crypt depth and area % of CD3+ and CD20+ cells. RESULTS: Horses on the butyrate supplemented diet had significantly reduced crypt depths in the right dorsal colon compared to placebo-fed horses (P < 0.001). However, a treatment x period interaction (P = 0.002) was discovered regarding this parameter, which could not be explained by the authors. Further investigation into the number of KI67+ cells in the RDC crypts did not reveal any significant differences between treatments (P = 0.650), indicating that the reduction in crypt depth in butyrate-fed horses could not be explained by a significant difference in cellular proliferation. Intestinal pH, villus length and expression of intestinal CD3+ and CD20+ cells were not significantly affected by treatment at any intestinal level. CONCLUSIONS: Our data indicate that supplementation of micro-encapsulated sodium butyrate to the equine diet did not influence gut histology (with the exception of a decrease found in the crypts of the RDC) or immunohistochemistry parameters in healthy horses. Further research is warranted to investigate the impact of butyrate supplementation in horses with intestinal disease.

Occlusal fissures in equine cheek teeth: µCT and histological findings.

Fissures on the occlusal surface of equine cheek teeth are commonly encountered during oral examination. Generally, their presence is considered abnormal but their aetiopathogenesis and clinical impact on pulp disease is still undetermined. The aims of this research were to study the extent of occlusal cheek teeth fissures using high-resolution X-ray computed tomography (µCT) imaging and their histological characteristics. Twenty-seven teeth (of 15 horses) were scanned using µCT imaging to analyse fissure extent. Histological examination of a subset of teeth (n=7) was performed. Forty-three type 1 fissures (35 type 1a, eight type 1b) were identified. The mean length of the fissure of type 1a and type 1b on the occlusal surface was 3.47+/-1.60mm and 13.64+/-7.40mm, respectively. Their mean depth was 13.22+/-10.76mm and 7.42+/-6.42mm, respectively. Potential risk factors associated with fissure depth were identified using a multivariable mixed model The location of the fissure and Triadan number were significantly associated with fissure depth. Fissures could be identified on histological sections with the presence of organic material inside the fissure, microorganisms in the continuation of the fissure extending into the dentinal tubules and the presence of reactionary dentine. This study suggests that fissures can provide a pathway for microorganisms to enter the dentinal tubules, potentially resulting in local pulpal inflammation. It appears that in healthy teeth, vital odontoblasts react by producing reactionary (tertiary) dentin, which lacks patent tubules and thus provides a proper seal.

Equine cheek teeth occlusal fissures: Prevalence, association with dental wear abnormalities and occlusal angles.

BACKGROUND: Fissures of the occlusal surface of equine cheek teeth are commonly encountered during oral examination but their aetiology is unknown. OBJECTIVES: To examine the prevalence and characteristics of occlusal fissures in cadaver teeth. It is hypothesised that their prevalence is influenced by masticatory forces. Consequently, their possible association with wear disorders and occlusal angles were examined. STUDY DESIGN: Cross-sectional survey. METHODS: The dental abnormalities and occlusal fissure findings in the cheek teeth of 143 cadaver heads were recorded. The cheek teeth occlusal angles were measured using the stiff-hinge technique. Multiple regression analyses were performed to establish possible relationships between age, sex, dental wear, occlusal angle and fissure prevalence. RESULTS: Occlusal fissures were found in 103/143 (72%) heads. Sex and age were determining factors in the prevalence of fissures. A similar prevalence was found in mandibular (54.1%) and maxillary teeth (45.9%, OR = 1.10; 95% CI = 0.95-1.29, P = 0.2). Mandibular fissures were more commonly located on the buccal aspect (OR = 1.42; 95% CI = 1.16-1.65, P < 0.001), whereas for maxillary fissures there was no difference in prevalence between palatal and buccal aspects (OR = 1.19; 95% CI = 0.97-1.46, P = 0.1). Two main fissure types were identified. Type 1a fissures were the most prevalent type (39.5%). No significant correlation was found between the presence of wear abnormalities or the occlusal angle of cheek teeth, and the prevalence of fissures. MAIN LIMITATIONS: No dental histories were available. CONCLUSION: Equine cheek teeth show a high prevalence of occlusal fissures. Despite some evidence of predilection sites on the tooth surface that might indicate a mechanical aetiology for these lesions, no associations were found with wear abnormalities or occlusal angles of affected cheek teeth. Further histological and ultrastructural studies are warranted to elucidate their aetiology and possible role in other dental diseases.

The use of a rein tension device to compare different training methods for neck flexion in base-level trained Warmblood horses at the walk.

BACKGROUND: Debate surrounds the use of high rein tension for obtaining different head and neck positions in the training of sport horses on account of possible welfare issues. OBJECTIVES: To compare auxiliary rein tension in two methods (Draw Reins and Concord Leader) for obtaining a standardised head and neck position on a hard and a soft surface. STUDY DESIGN: Intervention study. METHODS: Left and right rein tensions were measured in 11 base-level trained client-owned sport horses (mean age ± s.d.; 10 ± 3.2 years) exercised in-hand with, in a random order, conventional draw reins or the newly developed Concord Leader in a standardised head and neck position. Rein tension was measured using a calibrated device operating at 10 Hz during six runs of 15 s in a straight line for each training method on both a hard and a soft surface. A linear mixed model and grouped logistic regression analysis were applied to compare the two methods (P<0.05). RESULTS: The odds of a tension of 0 N were lower with draw reins than with the Concord Leader. The rein tension (mean sum of the force applied, in N) of the draw reins was 13.8 times higher than that of the Concord Leader. MAIN LIMITATIONS: This study was performed on horses exercised in-hand; however, these auxiliary aids are normally used when lungeing. Possible redirection of rein tension towards the poll was not measured. CONCLUSIONS: We showed that when using the Concord Leader a similar head and neck position is achieved with a much lower rein tension than with the draw reins and, more importantly, with a much greater likelihood of 0 N. It is unnecessary to use high auxiliary rein tension to obtain a standard, flexed head and neck position.

The possible role of Stomoxys calcitrans in equine sarcoid transmission.

The association between bovine papillomavirus (BPV) and equine sarcoids is well established, but it is unclear how the virus spreads. Although evidence in support of viral spread through direct animal contact exists, this does not explain sarcoid development in isolated equids. BPV DNA has been detected in flies, which could indicate that these insects serve as a vector. This study aimed to investigate whether BPV-negative stable flies (Stomoxys calcitrans) become positive for BPV DNA after exposure to equine sarcoid or bovine papilloma tissue under experimental conditions and, if so, for how long. A total of 420 stable flies were caught alive and exposed to BPV positive equine sarcoid or bovine papilloma tissue. During the following week, dead flies were collected daily and BPV loads were determined by quantitative PCR. There was a significant rise in BPV load after tissue exposure both in sarcoid and papilloma exposed flies, but the viral load was higher and remained high for a longer time after exposure to papilloma tissue compared to sarcoid tissue. Within days, viral loads decreased again and became indifferent from loads before exposure. The results of these experiments indicate that BPV transmission by S. calcitrans seems possible and is more likely to occur after contact with bovine papillomas than with equine sarcoids. Transmission seems only possible shortly after tissue exposure. Further research could include experimental induction of sarcoids with BPV positive stable flies, or a repeat of the experiment with micro-dissection prior to PCR.

Topical distribution of acyclovir in normal equine skin and equine sarcoids: An in vitro study.

Topical acyclovir application is an owner-friendly treatment for occult equine sarcoids, without the caustic side-effects other topical treatments have. Variable clinical success rates have been described, but it is not known to what rate and extent acyclovir penetrates in and through equine skin from a topical formulation. In the current study, an in vitro Franz diffusion model was used to determine the permeation parameters for a generic 5% acyclovir cetomacrogol cream for both healthy and sarcoid equine skin. The distribution of acyclovir between different layers of both skin types was also evaluated. While acyclovir penetrated through both skin types, significantly less acyclovir permeated to the deep dermis of sarcoid skin (197.62ng/mm(3)) compared to normal skin (459.41ng/mm(3)). Within sarcoid skin samples, significantly higher acyclovir concentrations were found in the epidermis (983.59ng/mm(3)) compared to the superficial dermis (450.02ng/mm(3)) and the deep dermis. At each sample point, significantly more acyclovir permeated to the receptor fluid through normal skin compared to sarcoid skin, which is reflected in the significantly higher permeation parameters of normal skin. Normal skin was found to be more permissive for acyclovir, but even in sarcoid skin, enough acyclovir reached the deep dermis to treat a Herpes simplex virus infection. In the case of equine sarcoids, the treatment is aimed at the Bovine papillomavirus and no information is available on the susceptibility of the DNA polymerase of this virus for acyclovir. Therefore, further research is needed to determine the efficacy of acyclovir to treat equine sarcoids.

Recording of cord dorsum potentials in calves.

Cord dorsum potentials are sensory evoked potentials being used to assess proximal sensory nerve, dorsal nerve root and spinal cord dorsal horn function. The purpose of the present study was to establish normal values for onset latency, peak latency and peak-to-peak amplitude of cord dorsum potentials after saphenous nerve stimulation in 15 healthy calves. Under general anesthesia, the saphenous nerve was stimulated in the periphery and cord dorsum potentials were recorded from the interarcuate space L3-L4, L4-L5, L5-L6 and L6-S1. Cord dorsum potentials were easily recorded at the different recording sites and consisted of a large negative peak followed by a long latency positive phase. The largest responses were recorded at the L5-L6 interarcuate space. Body temperature significantly influenced peak-to-peak amplitude. Onset latency prolonged with increasing limb length.

Enhancing bone healing and regeneration: present and future perspectives in veterinary orthopaedics.

Methods currently used to restore bone defects in human and veterinary orthopaedics are often not satisfactory. This is especially the case in the healing of large, irregular defects which result in the formation of tissues with inferior qualities compared to the original structures. For these reasons, several new approaches are currently being explored to improve bone healing capacities in different situations. This review will examine the different techniques used to enhance bone regeneration, highlighting both experimental and clinically applicable methods with regard to veterinary orthopaedics.

Distal limb cast sores in horses: risk factors and early detection using thermography.

REASONS FOR PERFORMING STUDY: There is a lack of evidence-based data on the prevalence, outcome and risk factors of distal limb cast sores, and no objective tool has been described for the early detection of cast sores. OBJECTIVES: To investigate the prevalence, location, outcome and risk factors of cast sores after application of a distal limb cast and to determine whether static thermography of the cast is a valuable tool for the assessment of sores. METHODS: A prospective study was conducted on horses treated with a distal limb cast. At each cast removal, cast sores were graded as superficial sores (SS), deep dermal sores (DS) or full thickness skin ulcerations (FS). In several cases, a thermographic evaluation of the cast was performed immediately prior to removal and differences in temperature (AT) between the coolest point of the cast and 2 cast regions predisposed for sore development (dorsoproximal mc/mtIII and palmar/plantar fetlock) were calculated. RESULTS: Mean +/- s.d. total casting time of 70 horses was 31 +/- 18 days. Overall, 57 legs (81%) developed at least SS. Twenty-four legs (34%) ultimately developed DS and one horse had an FS. Multivariable analysis showed that the severity of sores was positively associated with increasing age (OR: 1.111, P = 0.028), a normal (vs. swollen) limb (OR: 3387, P = 0.023) and an increase in total casting time (OR per week: 1.363, P = 0.002). The thermographic evaluation (35 casts) revealed that the severity of sores was positively associated with increasing deltaT (OR: 2.100, P = 0.0005). The optimal cut-off values for the presence of SS and DS were set at, respectively, deltaT = 23 and 43 degrees C. CONCLUSION AND POTENTIAL RELEVANCE: Distal limb cast is a safe coaptation technique with increasing risk of developing sores with time. Thermography is a valuable and rapid clinical tool to monitor the development of cast sores.

Cardiorespiratory effects of enoximone in anaesthetised colic horses.

REASONS FOR PERFORMING STUDY: No studies have been reported on the effects of enoximone in anaesthetised colic horses. OBJECTIVE: To examine whether enoximone improves cardiovascular function and reduces dobutamine requirement in anaesthetised colic horses. METHODS: Forty-eight mature colic horses were enrolled in this prospective, randomised clinical trial. After sedation (xylazine 0.7 mg/kg bwt) and induction (midazolam 0.06 mg/kg bwt, ketamine 2.2 mg/kg bwt), anaesthesia was maintained with isoflurane in oxygen and a lidocaine constant rate infusion (15 mg/kg bwt, 2 mg/kg/h). Horses were ventilated (PaCO2 < 8.00 kPa). If hypotension occurred, dobutamine and/or colloids were administered. Ten minutes after skin incision, horses randomly received an i.v. bolus of enoximone (0.5 mg/kg bwt) or saline. Monitoring included respiratory and arterial blood gases, heart rate (HR), arterial pressure and cardiac index (CI). Systemic vascular resistance (SVR), stroke index (SI) and oxygen delivery index (DO2I) were calculated. For each variable, changes between baseline and T10 within each treatment group and/or colic type (small intestines, large intestines or mixed) were analysed and compared between treatments in a fixed effects model. Differences between treatments until T30 were investigated using a mixed model (a = 0.05). RESULTS: Ten minutes after enoximone treatment, CI (P = 0.0010), HR (P = 0.0033) and DO2I (P = 0.0007) were higher and SVR lower (P = 0.0043) than at baseline. The changes in CI, HR and SVR were significantly different from those after saline treatment. During the first 30 min after enoximone treatment, DO2I (P = 0.0224) and HR (P = 0.0003) were higher than after saline administration. Because the difference in HR between treatments was much clearer in large intestine colic cases, an interaction was detected between treatment and colic type in both analyses (P = 0.0076 and 0.0038, respectively). CONCLUSIONS: Enoximone produced significant, but short lasting, cardiovascular effects in colic horses. POTENTIAL RELEVANCE: Enoximone's cardiovascular effects in colic horses were of shorter duration than in healthy ponies.

Virtual histology by means of high-resolution X-ray CT.

Micro-CT is a non-destructive technique for 3D tomographic investigation of an object. A 3D representation of the internal structure is calculated based on a series of X-ray radiographs taken from different angles. The spatial resolution of current laboratory-used micro-CT systems has come down over the last years from a few tens of microns to a few microns. This opens the possibility to perform histological investigations in 3D on a virtual representation of a sample, referred to as virtual 3D histology. The advantage of micro-CT based virtual histology is the immediate and automated 3D visualization of the sample without prior slicing, sample preparation like decalcification, photographing and aligning. This not only permits a drastic reduction in preparation time but also offers the possibility to easily investigate objects that are difficult to slice. This article presents results that were obtained on punch biopsies of horse skin, (dental) alveolus of ponies and chondro-osseous samples from the tarsus of foals studied with the new high resolution micro-CT set-up (HRXCT) at the Ghent University (Belgium) (http://www.ugct.ugent.be). This state-of-the-art set-up provides a 1 micron resolution and is therefore ideally suited for a direct comparison with standard light microscopy-based histology.

Immunohistochemical analysis of low-temperature methylmethacrylate resin-embedded goat tissues.

Goats are frequently used as a suitable animal model for tissue engineering. Immunohistochemistry can be helpful in improving the understanding and evaluation of the in vivo tissue responses at a molecular level. Several commercially available antibodies (KI67, vimentin, CD31, core-binding factor alpha-1, osteocalcin, alkaline phosphatase, MAC387, CD3, CD20, CD20cy, CD79 and CD45) were evaluated on Technovit 9100 New embedded goat tissues. Only vimentin, osteocalcin, MAC387 and CD3 revealed positive staining. These antibodies can be routinely used to evaluate goat tissues at molecular level. The use and development of alternative antibodies might further supplement and complete the possibilities for immunohistochemical analysis of goat tissue samples.

Evaluation of the in vivo behaviour of gentamicin sulphate ocular mini-tablets in ponies.

The in vivo behaviour of 5% gentamicin sulphate ocular mini-tablets (2-mm diameter, 6.525 mg weight) was compared with gentamicin eye drops in six ponies. Two mini-tablets were inserted on the bulbar conjunctiva of the right eye while a similar dose of gentamicin was administered via eye drops in the left eye. Irritation induced by the mini-tablets and the eye drops was evaluated using a visual analogue scale (0-10). Tears were sampled with ophthalmologic absorption triangles for 1 min for the determination of the concentration of gentamicin sulphate using a microbiological plate diffusion method. Irritation induced by the tablets was minor and clinically acceptable (overall median score of 1.7 +/- 1.4). Eye drops induced a sharp increase in gentamicin sulphate concentration (364.4 microg/mL after 5 min) followed by a fast decline (10.8 microg/mL after 60 min). The increase in concentration induced by the ocular mini-tablets was less pronounced (up to 56.2 microg/mL after 30 min) and followed by a gradual decrease; the concentration remained above 15 microg/mL for 8 h. Ocular 5% gentamicin sulphate mini-tablets are clinically well-tolerated in ponies, assuring a constant concentration in the tears for at least 8 h.

Evaluation of excision, cryosurgery and local BCG vaccination for the treatment of equine sarcoids.

Ninety-five horses with sarcoids were subjected to three types of treatment: surgical excision (conventional or carbon dioxide laser), cryotherapy or local BCG vaccination. The type of treatment was selected on the basis of the size, location and clinical appearance of the tumours. The choice between conventional and laser excision was empirical. A successful outcome was obtained in 11 of 14 (79 per cent) of the horses treated by cryosurgery, 18 of 27 (67 per cent) treated by BCG vaccination, 18 of 22 (82 per cent) treated by conventional excision, and 20 of 28 (71 per cent) treated with a carbon dioxide laser. For both excision methods, rigorous measures were taken to avoid autoinoculation and to ensure a wide margin of normal skin. The probability of local recurrence after excision was significantly higher for large sarcoids and sarcoids which had previously failed to respond to treatment. In 10 of the 31 horses with remaining sarcoids, some or all of the untreated sarcoids were observed to regress spontaneously.

Partial tibial neurectomy in 113 Belgian blue calves with spastic paresis.

One hundred and thirteen double-muscled Belgian blue calves (69 males and 44 females) with spastic paresis (52 unilateral and 61 bilateral) were treated surgically by partial tibial neurectomy under caudal epidural anaesthesia. Telephone inquiries made at least three-and-a-half months later established that good results were obtained in 83.2 per cent of the calves; a considerable improvement was reported in 4.4 per cent which still had intermittent spastic contractions; severe hyperflexion of the hock necessitating early slaughtering was recorded in 4.4 per cent of the calves; and in 8 per cent there was little or no improvement.

Histological study of the innervation of the suspensory ligament of the forelimb of the horse.

The innervation pattern of the interosseus muscle of the forelimb was studied in two ponies and two horses. The nerves of the suspensory ligament were studied histologically after neurectomy of the ulnar and median nerve branches proximal to the carpal joint. The results demonstrated that the interosseus muscle is innervated by the deep branch of the lateral palmar nerve which emerges at the level of the midcarpal region and contains fibres from the ulnar and the median nerve. These findings provide evidence that an ulnar nerve block proximal to the accessory bone would fail to anaesthetise the entire suspensory ligament.