Tissues Of Horses Research Articles

High Numbers of CD163-Positive Macrophages in the Fibrotic Region of Exuberant Granulation Tissue in Horses.

Simple SummaryHorses are prone to develop a wound healing disorder on their limbs called exuberant granulation tissue (EGT). The exact mechanism for the formation of this tissue remains unknown but the inflammatory response is supposed to be an important contributing factor. In this article, we investigated this inflammatory response in both EGT wounds as well as in control horse wounds. In biopsies, we detected two types of immune cells: (1) immune cells involved in early inflammation (MAC387+ cells) and (2) immune cells important in the later phases of inflammation (CD163+ cells). We detected a higher number of immune cells in EGT wounds compared with the control wounds of 19 days old. This suggests that EGT wounds may not be able to proceed through further phases in the wound healing process or that the inflammation phase is prolonged. Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase.

Isolation and morphological characterization of equine mesenchymal stem cells from harvested adipose tissue and bone marrow and stably transfected with green fluorescent protein.

To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characteristics of transfected and nontransfected AT-MSCs and BM-MSCs were compared with electron microscopy. The protocols for transfection and subsequent isolation of transfected cells with use of G418 were suitable for obtaining transfected MSCs. Transfection efficiency was 5% in AT-MSCs and 4% in BM-MSCs. Characterization of transfected and nontransfected MSCs revealed that they share immunocytochemical and morphological profiles. Expression of CD90 was significantly higher for transfected versus nontransfected AT-MSCs (97% vs 92%). Expression of CD105 was significantly lower for transfected versus nontransfected BM-MSCs (85% vs 94%). Transfected BM-MSCs had differences in organelles, compared with the other cell types, specifically including most commonly the rough endoplasmic reticulum with dilated cisternae and mitochondria. These findings contribute to the knowledge base of the characteristics of equine AT-MSCs and BM-MSCs and of transfected versus nontransfected equine MSCs. The data provided a valuable starting point for researchers wishing to further study the morphological characteristics of equine MSCs.

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 parvovirus hepatitis.

Equine parvovirus hepatitis (EqPV‐H) was first described in 2018 in a fatal case of Theiler's disease which followed the administration of an equine‐origin biological product. The virus has since been frequently identified in serum and liver tissue of horses affected by Theiler's disease—an acute, severe hepatitis characterised by fulminant hepatic necrosis with a fatal outcome in most cases. EqPV‐H is hepatotropic, appears to be associated with subclinical to severe hepatitis in horses, and is a likely cause of Theiler's disease. Although this disease is most frequently reported following the administration of equine‐origin biological products, it can also occur among in‐contact horses. Horizontal transmission may be iatrogenic, via contaminated equine‐origin biological products such as equine serum, botulism or tetanus antitoxin, and mesenchymal stem cells or by means of the oral route of infection. Other horizontal transmission routes, for example, arthropod vectors, warrant further investigation. A worldwide prevalence of EqPV‐H antibodies and DNA has been reported in asymptomatic horses. EqPV‐H‐positive horses suffering from acute, severe hepatitis have reportedly developed clinical signs including icterus, lethargy, inappetence, and neurological abnormalities and have had increased liver‐associated biochemistry parameters recorded. The most common histopathological abnormalities of the liver have been hepatocellular necrosis, collapse of the lobular architecture, and lymphocytic infiltration. Most horses infected experimentally with EqPV‐H have developed subclinical hepatitis, and close temporal associations between peak viraemia, seroconversion, and the onset of hepatitis have been observed. Based on strong evidence indicating that EqPV‐H causes hepatitis in horses, veterinarians should consider this virus an important differential diagnosis in such cases. Potential risks associated with the administration of equine‐origin biological products must be emphasised.

Speed and Blood Parameters Differ between Arabian and Žemaitukai Horses during Endurance Racing.

Simple SummaryLimited information exists on the physiological changes that occur in the horses competing in endurance races. The objective was to provide the initial data describing changes in laboratory measurements of the horses competing in endurance races under temperate conditions and to compare the data between the Arabian horses, which are one of the most popular horse breeds in the world, and a Lithuanian horse breed—Žemaitukai. The study was carried out on 112 horses. Blood samples were collected before and after an endurance race. The Arabian horses were faster compared to the local breed (Žemaitukai). The study showed significant changes in horse blood gasometrical and biochemical indicators.Fédération Equestre Internationale (FEI) has described equine endurance racing as the second largest discipline in the world, above which is only show jumping. The Žemaitukai is an ancient indigenous Lithuanian horse breed known since the 6th or 7th century. The Arabian horse breed is one of the oldest human-developed horse breeds in the world. Compared with other race horse breeds, the muscle tissue of Arabian horses is characterized by significant differences in structure—a predominance of oxidative fiber type I is observed in Arabians, making them the prevailing breed in endurance racing. The Arabian horses are recognized as the leading breed in endurance competitions. Speed, pace, and total time in the race strategy have been extensively studied in human sports, and in contrast, this strategy appears to have been virtually ignored in equestrian sport, despite the potential for contributing to performance optimization. In relation to speed and total time in the race, there are limited data on postrace physical, biochemical, and blood gas parameters of endurance horses. Thus, this study was carried out to investigate the effects of speed on the blood parameters of the Arabian and Žemaitukai horses during an endurance race. Blood samples were taken before and immediately after the exercise. Biochemical and blood gas indicators were analyzed. The study showed significant increases in mean blood gasometrical indicators, such as partial carbon dioxide pressure (8.09–15.18%, p < 0.001); base excess in the extracellular fluid (14.01%, p < 0.001 in the Arabian horses and 172.01% in the Žemaitukai breed, p = 0.006); decreases of the blood electrolyte ionized calcium (4.38–8.72%, p < 0.001) and the hematocrit and hemoglobin values (20.05–20.12%, p < 0.001 in the Arabian horses and 6.22–6.23% in the Žemaitukai breed, p = 0.003–0.004); and decreases in the base excess in the blood values (29.24–39.38%, p < 0.001) and lactate (13.45–31.97%, p < 0.001) in the blood of both breeds in the post-competition horses. Significant increases after competition were determined for the values of creatinine (21.34–30.82%, p = 0.001–0.004), total bilirubin (50.84–56.24%, PH < 0.001), and albumin (2.63–4.48%, p = 0.048–0.001) for both breeds. For the faster Arabian horse breed, recovering after racing took half the time that the local Žemaitukai breed did.

RADIOGRAPHIC, VENOGRAPHIC AND HOOF GROWTH EVALUATIONS IN EQUINE FORELIMBS WITH CHRONIC LAMINITIS TREATED OR NOT WITH MESENQUIMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE

<h3>Background</h3> The objective was to evaluate the effects of regional perfusion of characterized mesenchymal stem cells derived from adipose tissue (Ad-MSC) in horses with chronic laminitis. <h3>Methods</h3> Six mares with stable chronic laminitis in standard treatment for distal phalangeal realignment were subjected to regional perfusion of the digital vein with Ad-MSC (20 million cells, 3 infusions every 15 days) in one forelimb compared to placebo infusion in the contralateral one. Evaluations occurred in two 30-day periods, before and after treatment (from the second infusion). Radiographs and digital venographs were performed on both thoracic limbs at the beginning and end of each period. Venography was evaluated by a vascular perfusion score system. The growth of the hoof capsule was monitored by the distance between the coronary band and a radiopaque marker inserted in the dorsal hoof wall. Radiographic spatial relationship between the distal phalanx and the hoof capsule was assessed by a specific software (Hoof Metron). <h3>Results</h3> Compared to baseline values, vascular perfusion was improved only in the Ad-MSC group (P <0.03), with emphasis on perfusion of the lamellar dorsal vessels (P <0.03) and the circumflex-lamellar junction (P <0.03). There was hoof growth in both groups, but the mean final distance in the Ad-MSC limbs was 20% greater (P <0.03) than in placebos limbs. Positive effects were also detected in the spatial relationship between the distal phalanx and the hoof capsule of limbs treated with Ad-MSC. <h3>Conclusion</h3> Ad-MSC therapy promoted improvements in digital blood perfusion, growth rate, and spatial relationship between distal phalanx and hoof capsule. Our results confirm the therapeutic potential of Ad-MSC in the treatment of horses with stable chronic laminitis. Improvements only in treated hooves reveals that infusions must be performed regionally in each of the affected limbs.

LAMELLAR HISTOLOGICAL AND IMMUNOHISTOCHEMICAL EVALUATION OF THE HOOF WALL IN HORSES WITH CHRONIC LAMINITIS TREATED WITH MESENQUIMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE BY REGIONAL PERFUSION

<h3>Background</h3> The objective was to evaluate the histological and immunohistochemical effects of regional perfusion of characterized mesenchymal stem cells derived from adipose tissue (Ad-MSC) in biopsy hoof samples from horses with laminitis. <h3>Methods</h3> Six mares with stable chronic laminitis in standard treatment for distal phalangeal realignment were subjected to regional perfusion of the digital vein with Ad-MSC (20 million cells, 3 infusions every 15 days) in one forelimb compared to placebo infusion in the contralateral one. Evaluations occurred in two 30-day periods, before and after treatment (from the second infusion). Dorsal hoof lamellar biopsies were taken 30 days before and 15 days after the therapeutic protocols. Histological 5-μm cuts stained with H&E and PAS were classified based on a crescent 1-5 scale lesion score system. The sum of each lesion grade represented the global lesion score. Immunohistochemistry for p63 positive cells were also performed in formalin-fixed samples. <h3>Results</h3> In the placebo limbs, there were increases over time in following histological variables: global lesion (P=0.01), keratinized axis loss (P=0.0001), stratum medium hyperplasia (P=0.001), primary epidermal lamellae homogeneity loss (P=0.003), and hyperkeratosis (P=0.05). Only the stratum medium hyperplasia score was increased in the Ad-MSC-treated hooves. P63-positive cells could be detected in all formalin-fixed paraffin-embedded samples, but background unspecific stain made counts imprecise to be made. <h3>Conclusion</h3> In conclusion Ad-MSC regional vascular perfusion prevented histological lesion progression in the lamellar tissue of horses with chronic laminitis. Our results confirm the therapeutic potential of Ad-MSC in the treatment of horses with stable chronic laminitis.

VIABILITY AND DISTRIBUTION EVALUATION OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE MARKED BY NANOCRYSTALS IN HORSES WITH CHRONIC LAMINITIS 24 HOURS AFTER REGIONAL INFUSION - PRELIMINARY STUDY

<h3>Background</h3> The objective of this study was to evaluate the viability, distribution and setting of previously characterized and differentiated equine Ad-MSC labeled with the Qtracker cell labeling kit after infusion into the forelimb of horses with chronic laminitis. <h3>Methods</h3> Subcutaneous fat from healthy horse was collected, and Ad-MSC were isolated and cultured. In the third pass, Ad-MSC were marked with fluorescent nanocrystals. Labeling was carried out according to the manufacturer‘s instructions. Briefly, two million Ad-MSC were marked by adding 2ul of nanocrystals, 2ul of PBS and 200ul of DMEM medium, followed by incubation at 37°C and 5% CO₂ for 1 hour. Cell viability was determined by staining with trypan blue before staining and at 1 and 24 hours after staining. 20 million allogeneic Ad-MSC marked according to the manufacturer‘s standards and suspended with 20 ml of PBS were infused in 2 horses with chronic laminitis. The infusion was performed only in one member (treated), previously drawn, by regional limb perfusion (RLP) and the tourniquet was maintained for 30 minutes. In the contralateral limb, only PBS was infused. Lamellar tissue biopsies were performed 24 hours later. The absorption of nanocrystals in the cell was observed through fluorescence microscopy in samples of labeled cells and lamellar tissue obtained by biopsy. <h3>Results</h3> Cytoplasmic absorption of nanocrystals was observed 1 and 24 hours after staining. The viability of Ad-MSC in culture before labeling was 91%. After 1 hour of incubation with nanocrystals, viability decreased slightly to 85% followed by 83% viable cells 24 hours after incubation. The results of this study demonstrate that Qdot nanocrystals can be successfully introduced into equine Ad-MSC with minimal cytotoxic effect on these cells. The cytoplasmic absorption of the nanocrystals was observed in the form of cell niches in the secondary laminae in both animals, but in greater quantity in the samples of the treated limb. Although lamellar tissue autofluorescence was observed in both limbs, there was a wide predominance of labeled Ad-MSC in the treated limbs. <h3>Conclusion</h3> The cell labeling protocol was successful, and the labeled cells remained in the lamellar tissue of horses with chronic laminitis 24 hours after the RLP. The preliminary findings of this study indicate that the cells remained in the limb that received the RLP, demonstrating therapeutic potential in the use of this technique.

A comparison of haematological and biochemical blood indices between the Žemaitukai and Arabian horses participating in endurance competitions

The study was conducted on 30 clinically healthy Arabian horses and 28 Žemaitukai horses that competed in endurance race over the same distance (40 km). Blood samples were taken before and immediately after the exercise. The environmental conditions during the competitions varied, with a mean temperature of 22.5 °C and a mean relative humidity of 73.20%. The Žemaitukai horses showed lower haematological values and increased muscle catabolism after exercise. Arabian horses have higher aerobic capacity compared to the Žemaitukai horses which probably contributes to their superior low- to moderate-intensity exercise performance. Serum activity of muscle enzymes suggested that the muscle tissue of the Arabian horses has higher tolerance for exercise-induced muscle catabolism and lower muscle catabolism than that of muscle tissue of the Žemaitukai horses. Further studies need to be carried out to identify the diferences in muscle tissue of both breeds.

Gene Expression Profile in Similar Tissues Using Transcriptome Sequencing Data of Whole-Body Horse Skeletal Muscle.

Horses have been studied for exercise function rather than food production, unlike most livestock. Therefore, the role and characteristics of tissue landscapes are critically understudied, except for certain muscles used in exercise-related studies. In the present study, we compared RNA-Seq data from 18 Jeju horse skeletal muscles to identify differentially expressed genes (DEGs) between tissues that have similar functions and to characterize these differences. We identified DEGs between different muscles using pairwise differential expression (DE) analyses of tissue transcriptome expression data and classified the samples using the expression values of those genes. Each tissue was largely classified into two groups and their subgroups by k-means clustering, and the DEGs identified in comparison between each group were analyzed by functional/pathway level using gene set enrichment analysis and gene level, confirming the expression of significant genes. As a result of the analysis, the differences in metabolic properties like glycolysis, oxidative phosphorylation, and exercise adaptation of the groups were detected. The results demonstrated that the biochemical and anatomical features of a wide range of muscle tissues in horses could be determined through transcriptome expression analysis, and provided proof-of-concept data demonstrating that RNA-Seq analysis can be used to classify and study in-depth differences between tissues with similar properties.

Effect of Caudal Traction on Mechanical Nociceptive Thresholds of Epaxial and Pelvic Musculature on a Group of Horses With Signs of Back Pain

Direct muscular attachment from lumbar vertebrae to the caudal vertebrae of the tail suggests that caudal traction, also described as a tail pull, may affect lumbar vertebral segments and/or associated soft tissues in horses. Traction is a commonly used human manual therapy technique used for pain relief and anecdotally observed to relieve pain in horses. However, research is lacking validating the efficacy of manual caudal traction on the horse. The objective of this study was to determine if caudal traction has an effect on mechanical nociceptive thresholds (MNTs) in a group of horses with clinical signs of back pain. Pressure algometry was used to measure MNTs of five bilateral anatomical sites in the epaxial and pelvic musculature of 11 horses referred to physiotherapy because of clinical signs of back pain. Measurements were recorded both before and immediately after traction. A significant difference (P ≤ .05) was identified between mean before and after caudal traction algometry measurements in all described sites. The percentage of MNT increase was highest in the thoracic region (83%) compared with the lumbar (50%) and the pelvic (52.4%) regions. These results support an effect of caudal traction in increasing MNTs in the thoracolumbar and pelvic regions in horses. Further research to determine the clinical effect of this technique is warranted.

Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept)

BackgroundThere is a growing interest in the scientific community to use computer-based software programs for the quantification of cells during physiological and pathophysiological processes. Drawbacks of computer-based methods currently used to quantify immunohistochemical staining are the complexity of use, expense of software and overly-simplified descriptions of protocol thereby limiting reproducibility. The precise role of mast cells in equine cutaneous wound healing is unknown. Given the contribution of mast cells to the chronic inflammation observed in human keloid, a pathology similar to exuberant granulation tissue (EGT) in horses, mast cells might be present in high numbers in equine limb wounds predisposed to EGT. The main goal of this study was to develop a reliable and reproducible quantification method for immunostained tissues using a computer software that is widely available, at no cost, to the scientific community. A secondary goal was to conduct a proof of concept using the newly-established method to quantify mast cells during wound healing at different anatomical sites (body and limb) in horses to see if a different pattern is observed in limb wounds, which are predisposed to EGT.ResultsA good intraclass correlation coefficient (ICC, 0.67 p < 0.05) was found between the computer-based ImageJ method and manual counting. An excellent intra-operator ICC of 0.90 (p < 0.01) was found for the ImageJ quantification method while a good interoperator ICC of 0.69 (p < 0.01) was measured. No significant difference was observed between the variation of the ImageJ and that of the manual counting method. Mast cells were localized below the epidermis, around cutaneous appendages and blood vessels. Mast cell numbers did not differ significantly in relation to anatomical location or time of healing.ConclusionsThe computer-based quantification method developed is reliable, reproducible, available, cost-free and could be used to study different physiological and pathological processes using immunohistochemistry.

The transfer of 239+240Pu, 241Am, 137Cs and 90Sr to the tissues of horses

Horses are important food sources in several countries however, data on their radionuclide uptake is less available than for many other farm animals. Information on the transfer of artificial radioisotopes from the environment to the food supply is necessary for internal dose assessment and assuring the safety of the population relying on this food source. This study provides data for a less studied farm animal and, in the case of 241Am and 239+240Pu, relatively poorly studied radionuclides with respect to transfer to animal products. The transfer parameters for 239+240Pu, 241Am, 137Cs and 90Sr to the organs of 1-year old fillies, 10-year old mares and through the placental barrier into foetuses were quantified after 60-days feeding with contaminated soil or diet contaminated by a leachate solution. The transfer of radionuclides from ingested soil to tissues was generally lower, by up to three orders of magnitude, than from a diet contaminated by a leachate solution. The ingestion of soil is a particularly important source of radionuclide intake to grazing animals in the Semipalatinsk Test Site. For 241Am there is a lack of available data, the two singular entries for mutton and beef in the IAEA handbook are higher than all values observed in the current study. The maximum observed transfer factor for 241Am was 72 ± 22*10−5 d kg−1 FW in the liver of the mare fed with leachate contaminated feed. For 239+240Pu the maximum transfer factor was 31.8 ± 8*10−5 d kg−1 FW observed also in the liver of the mare fed with leachate contaminated feed. The filly fed with leachate contaminated feed had the highest transfer parameter value for 137Cs, 35.3*10−3 d kg−1 FW. The highest 90Sr transfer factor was found in the ribs of the filly fed leachate contaminated feed, 720 ± 144 *10−3 d kg−1 FW. The results presented in this paper can be used to improve the current internal dose estimates from the ingestion of horse meat produced in the area, however they are based on a low sample size; future studies need to use a larger number of animals.

First report of equine parvovirus-hepatitis-associated Theiler's disease in Europe.

Equine parvovirus-hepatitis (EqPV-H) has been proposed as the aetiological cause of Theiler's disease, also known as serum hepatitis. EqPV-H-associated Theiler's disease has not been previously reported in Europe. To determine whether EqPV-H infection was associated with a 2018-2019 outbreak of Theiler's disease in four horses on a studfarm. Descriptive case series. The medical records of four horses from the same farm diagnosed with fatal Theiler's disease were examined retrospectively. Information collected included a clinical history, physical examination findings, tetanus antitoxin exposure, serum biochemistry and necropsy reports. Liver tissue from all four horses was tested for EqPV-H using PCR and in situ hybridisation (ISH) assays. Three of the horses had a history of recent (7-11weeks) tetanus antitoxin administration. Liver tissue from all four horses tested positive for EqPV-H with PCR. In situ hybridisation revealed a widespread distribution of viral nucleic acid in hepatocytes in one case, and a more sporadic distribution in the remaining three cases. Case controls were not available from the farm in question given the retrospective nature of analysis. This case series documents the first reported EqPV-H-associated Theiler's disease in Europe and the first use of ISH to visualise the viral nucleic acid in liver tissues of horses with Theiler's disease.

First Report of Equine Leishmaniosis in the State of Sergipe, Brazil

Background: Leishmaniasis is caused by an obligate intracellular protozoan of the genus Leishmania. In Brazil, the species Leishmania (Leishmania) infantum is reported as an etiological agent of leishmaniasis. Transmission of the disease occurs through bite of the sand fly mosquitoes Lutzomyia longipalpis and Lutzomyia cruzi, present in several regions of Brazil, including the state of Sergipe, where they are considered endemic. Although few cases have been reported in other states of Brazil, no case of cutaneous leishmaniasis in horses has been reported in Sergipe. The objective of this study was to report a case of cutaneous leishmaniasis in a horse in the municipality of Itaporanga d'Ajuda, Sergipe, Brazil.Case: A 2-year-old female quarter-horse was examined by a private veterinarian due to the presence of a nodular lesion on the mucocutaneous region of the lips. Macroscopically, the nodule was alopecic with depigmented areas, measuring approximately 1.0 cm in diameter, and there was a focal ulcer in the region of the labial commissure. Excisional biopsy was performed as follows; the material obtained was fixed in 10% buffered formalin and sent for histopathological examination. Further, it was subjected to routine histological processing by embedment in paraffin and hematoxylin-eosin staining. Microscopically, there were intense multifocal to coalescent lymphohistioplasmocytic infiltrates with epithelioid macrophages and multinucleated giant cells, both containing amastigote forms comparable with Leishmania spp. Morphological diagnosis was defined as extensive multifocal to coalescent chronic granulomatous dermatitis with intracytoplasmic amastigote forms. Furthermore, for positive controls, immunohistochemical examination was performed using serum from dogs naturally infected with L. (L.) infantum, which resulted in a strongly positive immunostaining of innumerable intracytoplasmic amastigote structures, confirming infection by Leishmania infantum.Discussion: Leishmaniasis is a public health problem and is frequently studied. In the biological cycle of the parasite, canids are considered as reservoirs, and humans are considered as accidental hosts. However, in endemic rural areas, several species of mammals can be infected. Microscopic lesions of cutaneous leishmaniasis are compatible with granulomatous dermatitis, characterized by lymphohistioplasmocytic inflammatory infiltrates with epithelioid macrophages containing amastigote forms. Age, breed, and sex are not considered predisposing factors for this disease and their transmission depends on the existence of the vector in the environment. The diagnosis is based on serological, molecular, and immunohistochemical techniques; in the present study, immunohistochemistry was performed for diagnostic confirmation using serum from dogs naturally infected with L. (L.) infantum. It was found that this antibody can be successfully applied in the immunohistochemical diagnosis of leishmaniasis in tissues of horses. Histopathological examination including immunohistochemical analysis was essential to confirm the diagnosis. To the best of our knowledge, this is the first report of Leishmania infantum infection in a horse in the state of Sergipe, Brazil. It is essential to carry out epidemiological studies to define control measures to be implemented in the state since it is important for public health.

Prevalence and severity of ultrasonographic pulmonary findings in horses with asthma - a preliminary study.

Asthma is one of the most common non-infectious respiratory diseases in horses. Ultrasound examination is a widely available non-invasive additional diagnostic tool. To date, there are no studies focusing on ultrasonographic findings in horses with asthma. The aim of this study was to analyse the prevalence and severity of ultrasound lesions in lung tissue in horses with asthma. Lung ultrasonography was carried out on six healthy horses (controls) and 12 horses with asthma (six with mild and six with severe asthma). The sonographic changes in three lung sections were assessed using a scoring system. The most common changes present in all the animals were comet- tail artefacts. More advanced lesions were present in horses with severe asthma. Statistically significant differences in the overall average intensity of the ultrasound changes were seen between the controls and the study group and between the horses with mild and severe asthma. The lesions were usually located in the caudal lung regions, but they were also present in other areas as the disease progressed. Ultrasonography is a useful additional diagnostic tool enabling an assessment of the stage of the asthma progression. It is a very sensitive technique that visualizes minor lesions in the lung tissue even in clinically healthy animals. Due to its low specificity, it cannot replace endoscopy and the bronchoalveolar lavage in horses with asthma.

Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses

A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10% unbuffered formalin. Two deparaffinization methods and three proteinase K-based lysis step were tested and translated into three protocols. After detailed statistical analysis it was determined that a longer period and increase in volume of proteinase K incubation provide higher yields and purity of RNA (P < 0.01) of archived samples. Alongside, amplification of equid-housekeeping gene up to 298 bp was successful with the protocol adaptations. For different formalin-fixation timepoints, it was demonstrated that the right choice for treatment and formalin-fixation period is organ-related (P ≤ 0.05). Essentially, little alterations to pre-existing extraction protocols unwound the RNA of up to 11-year-old samples, enabling the use of FFPE tissue archives or e.g. harshly fixed material needed in infection research under high biosafety levels for a variety of molecular analysis.

Use of triamcinolone as treatment of granulation tissue (Proud flesh) in horses: case report

The occurrence of wounds and traumas in horses is relatively frequent of those animals. Because of having a great reserve of energy, combined with the agility and the instinct to run or fight, besides other factors, those animals are more predisposed to the occurrence of wounds. The management of wounds in horses is relatively delicate, since complications are not uncommon, and it can delay the treatment, as well as raise costs and require special care. Some points should be observed when handling wounds in horses, such as complete epithelialization without scar formation; shorter treatment time; avoid recurrence or complications that prolong the treatment, reduce costs. Exuberant granulation tissue is a common complication that occurs during the management of wounds in horses, and may be caused by prolonged treatment, wounds in regions with more moviments, infections, among other factors. Currently, there are several forms of treatment of exuberant granulation tissue in horses.The objective of this study was to report the success of the topical use of triamcinolone in three horses that presented exuberant granulation tissue growth. The horses presented exuberant granulation tissue growth in the distal region of the thoracic limb after trauma that generated the lesion. Treatment with 1mg triamcinolone ointment was started as an attempt to control and reduce the granulation tissue and allow the wound to heal completely and it was effective in all three treated cases, since the granulation tissue was reduced and the wound healed without major scars or any other damage to the horse.

Особенности репродукции вируса болезни Борна в клетках головного мозга и лабораторная диагностика инфекции

The Borna Disease Virus (BDV) is a non-segmented RNA-containing virus belonging to the Bornaviridae family. The pathogen causes progressive meningoencephalitis in animals of various types. Despite numerous studies, some stages of reproduction of Bornavirus remain poorly understood, in particular, receptor-mediated penetration, retrograde transport into the nucleus, assembly and release of the virion, etc. The present report presents the results of demonstrating studies that were aimed at following the immunoenzyme method, the distribution of the main protein BDV ― phosphoprotein P (p24) in the brain cells of naturally susceptible animals, compare nnye data with the results of a number of authors, made mainly in experimental models. Microscopic examination of histological sections of the brain tissue of horses and sheep treated with specific serum against p24 BDV in a light or luminescent microscope observed a specific color in the form of: small granules; diffuse fluorescence of the cytoplasm; larger granules, apparently due to aggregation of endosomes, for axon transportation; formations in the form of beads, demonstrating the transport of viral material along the axon. In vitro results were obtained indicating that it is possible to transport RNPs via a short path through cytoplasmic bridges. The presented results will allow a better understanding of the neuropathogenesis of Born's disease and improve the diagnosis of the disease.

Fatty acid composition of mesenteric, cardiac, abdominal, intermuscular, and subcutaneous adipose tissues from horses of three body condition scores

Body condition score estimates overall adiposity in horses but not fatty acid (FA) composition of fat depots, which is associated with many biological functions and important for horse health. The objectives of the current study were to determine the FA composition of mesenteric (MS), cardiac (CD), abdominal (AB), intermuscular (IM), and subcutaneous (SC) adipose tissues in stock-type horses and identify their relationships with BCS. Twenty-one stock-type horses (BCS 4, n = 7, 468 ± 13 kg; BCS 5, n = 10, 455 ± 11 kg; BCS 6, n = 4,493 ± 12 kg) were humanely euthanized based on 3 primary criteria: geriatric, crippled, and/or unsafe. Fat tissues including MS around the small intestines, CD at the pericardium, AB from abdominal fat located inside the left flank, IM underneath the latissimus dorsi muscle in the thoracic region, and SC on the surface of semitendinosus muscle were collected within 30 min post-mortem, trimmed of connective tissues and muscles, frozen in liquid nitrogen, pulverized into fine powder, and stored at –80 °C. Fatty acids were determined by gas chromatography and expressed in percentage of total FA concentration. Influences of BCS (P 〈 0.05) and fat depot (P < 0.05), but not BCS × fat depot interaction (P > 0.05), were found for most FA, indicating that most FA were independently influenced by BCS and anatomical locations. However, some FA, such as 16:1n7, were influenced by BCS differently in various fat depots (PBCS×fat depot = 0.043). The major FA were palmitic (24.85%), stearic (5.54%), oleic (27.34%), linoleic (11.20%), and linolenic (17.95%) acids. Degree of saturation was not affected by BCS (P = 0.212). Most saturated depots were AB and CD (P ≤ 0.049). In MS, IM, and SC, PUFA percentage was smallest (P < 0.001), but SFA and MUFA percentages were similar (P ≥ 0.505). In CD, SFA and PUFA percentages did not differ (P = 0.740), but MUFA percentage was less than both (P < 0.001). Percentages of SFA, MUFA, and PUFA in AB all differed (P ≤ 0.008) with SFA being greatest. Body condition score influenced FA composition of equine adipose tissues but to a lower extent than the type of fat depots.