Horse Blood Research Articles

Identification of Differentially Expressed Genes after Endurance Runs in Karbadian Horses to Determine Candidates for Stress Indicators and Performance Capability.

RNA sequencing makes it possible to uncover genetic mechanisms that underlie certain performance traits. In order to gain a deeper insight into the genetic background and biological processes involved in endurance performance in horses, the changes in the gene expression profiles induced by endurance runs over long (70 km) and short (15 km) distances in the blood of Kabardian horses (Equus caballus) were analyzed. For the long-distance runs, we identified 1484 up- and 691 downregulated genes, while after short-distance runs, only 13 up- and 8 downregulated genes (FC > |1.5|; p < 0.05) were found. These differentially expressed genes (DEGs) are involved in processes and pathways that are primarily related to stress response (interleukin production, activation of inflammatory system) but also to metabolism (carbohydrate catabolic process, lipid biosynthesis, NADP metabolic process). The most important genes involved in these processes therefore represent good candidates for the monitoring and evaluation of the performance of horses in order to avoid excessive demands when endurance performance is required, like ACOD1, CCL5, CD40LG, FOS, IL1R2, IL20RA, and IL22RA2, on the one hand, and, on the other hand, for assessing the suitability of a horse for endurance races, like GATA2, GYG1, HIF1A, MOGAT1, PFKFB3, PLIN5, SIK1, and STBD1.

Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment.

Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as p-phenylenediamine (p-P), o-dianisidine (o-D) and, most recently, ammonium iron(II) sulfate (AIS). Once developed in humans, these assays are often used in veterinary medicine without appropriately optimizing in the animal species of interest. In this study, two assays using AIS and o-D as substrates have been compared and validated for Cp oxidase activity assessment in horse's plasma. The optimization of the assays was performed mainly by varying the buffer pH as well as the buffer and the substrate molar concentration. Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent Cp inhibitor. In the o-D assay, 500 µM sodium azide treatment completely inhibits the enzymatic activity of Cp, whereas, using the AIS assay, a residual analytical signal was still present even at the highest (2000 µM) sodium azide concentration. Even though the analytical values obtained from these methods are well correlated, the enzymatic activity values significantly differ when expressed in Units L-1. A disagreement between these assays has also been detected with the Bland-Altman plot, showing a progressive discrepancy between methods with increasing analytical values.

Impact of Acidulants on Salmonella and Escherichia coli O157:H7 in Water Microcosms Containing Organic Matter.

As demands for fresh water become more competitive between the processing plant and other consumers of water such as municipalities, interest has grown in recycling or reusing water for food processing. However, recycling the processing water from a poultry plant, for example, represents challenges due to increased organic loads and the presence of bacterial contaminants including foodborne pathogens. The objective in the current study was to evaluate the inactivation of Salmonella and E. coli O157:H7 using combinations (0.5% and 1%) of sodium bisulfate (SBS) and 1% lactic acid (LA) in water and water with organic matter in the form of horse blood serum (0.3%) with exposure times of 1 min and 5 min. Pathogen reductions after a 5 min exposure time were greater than corresponding reductions after a 1 min exposure time for all acid solutions. The Salmonella counts were significantly reduced (i.e., ≥1 log-unit) in all acid solutions after a 5 min exposure time with the combination of LA + SBS acid solutions being more effective than the corresponding 2% LA solutions. None of the acid solutions were effective in reducing the E. coli O157:H7 after a 1 min exposure time. The 1% LA + 1% SBS solution was the most effective acid solution against both pathogens and was the only acid solution effective in reducing E. coli O157:H7 by at least one log unit after 5 min of exposure.

Relationship between selenium, copper, zinc and their biomarkers in blood and skeletal muscle tissue in adult horses from southern Chile.

Microminerals are necessary for all bodily functions. In animal species, selenium (Se), copper (Cu) and zinc (Zn) all form part of antioxidant enzymes. Micromineral deficiencies, particularly Se, are well recognized in large animal species in Chile. Glutathion peroxidase (GPx) is a widely used biomarker for Se nutritional status and to diagnose Se deficiency in horses. Superoxide dismutase (SOD) is a Cu and Zn-dependant antioxidant enzyme, although it is not commonly used as a proxy for the nutritional status of these minerals. Ceruloplasmin (CP) is used as a biomarker of Cu nutritional status. This study´s objective was to explore correlation between said minerals and biomarkers in adult horses from southern Chile. Se, Cu, Zn, GPx, SOD and CP were measured in whole blood of a group of 32 adult horses (5-15 years old). Further, a second group of 14 adult horses (5-15 years old) underwent gluteal muscle biopsies to determine Cu, Zn, GPx and SOD. Correlations were determined by means of Pearson´s r coefficient. Significant correlations were found for blood GPx and Se (r= 0.79), blood GPx and SOD (r= -0.6), muscular GPx and SOD (r= 0.78), and Cu and CP (r= 0.48). These results confirm previously described strong association between blood GPx and Se in horses, validating the former´s use as a diagnostic proxy of Se deficiency in Chilean horses, and suggest significant interactions between GPx and SOD in blood and muscle tissue.

EPIZOOTIC SITUATION OF HEMOPARASITIC DISEASES OF FARM ANIMALS IN IRKUTSK REGION

Background. Cattle anaplasmosis and babesiosis and theileriosis of horses are a group of natural focal vector-borne diseases that cause significant economic damage to livestock and horse breeding around the world. On the territory of the Baikal region these diseases and their pathogens remain little explored.&#x0D; Purpose – to study the epizootic situation of blood-parasitic diseases of farm animals in the Irkutsk region, to establish the species affiliation of the identified pathogens and their genetic diversity.&#x0D; Materials and methods. For the presence of Babesia spp./Theileria spp. and Anaplasma spp. has been researched659 horse blood samples, 579 sheep blood samples, 25 goat blood samples and 647 cattle blood samples. For the detection of babesia and theileria, nested two-round PCR with primers from the 18S rRNA gene region was used, and for the detection of anaplasma DNA with primers from the 16S rRNA gene region. For study the genetic diversity of the identified pathogens, selective sequencing of samples was carried out with subsequent phylogenetic analysis.&#x0D; Results. The DNA of Anaplasma spp. was detected in blood samples of small ruminants from 12 districts of the region. The infection rate of sheep and goats was 68.7±1.9% and 68%±9.3%, respectively. Nucleotide sequences of the 16S rRNA gene fragment were determined in 68 A. ovis samples from the blood of sheep and goats. The researched sequences were conservative for the studied gene and differed from each other in two heterozygous sites of the gene(T/C and G/A nucleotide substitutions).They corresponded to the sequences of the prototype strain Haibei (CP015994), as well as to the A. ovis sequences previously found in the blood of sheep from Altai (Russia) and Mongolia, deer and D. niveus and D. nuttalli ticks from China.&#x0D; DNA of Babesia spp./Theileria spp. found in blood samples of horses from 13 districts of the region. The average infection of horses was 64.2%±1.9%. Two etiological agents of equine piroplasmosis were found in the blood of the examined horses: T. equi (genetic groups A and E) and B. caballi. Definite sequences of the B. caballi 18S rRNA gene (700–1146 bp) were identical to each other and differed by at least ten substitutions and one deletion from the sequences available in the GenBank database. However, the greatest similarity was observed with B. caballi sequences found in the blood of a horse from Brazil (KY952238), ticks from Kazakhstan (MN907451) and China (MN173021).&#x0D; The results indicate that DNA of Anaplasma and Babesia in the blood of cattle have not been identified.&#x0D; Conclusion: The data received evidence a wide distribution of foci of small cattle anaplasmosis and piroplasmosis of horses in the Irkutsk region and the need to continue monitoring their epizootic condition.

Regulatory T lymphocytes and selected cytokines in horses with mild to severe equine asthma and different inflammatory cytology profiles

This study determined Tregs and inflammatory cytokines in BALF and peripheral blood (PB) of adult horses with mild and severe asthma and different BALF cytological inflammation profiles. Horses of diverse breeds with asthma (age range: 2–20 years, n = 24) were divided into groups according to the number of points obtained in a standardized clinical scoring system (mild-moderate equine asthma – MEA, severe equine asthma – SEA) and according to the inflammation type based on cytological finding. Plasma levels of IFN-γ, IL-4, IL-10, IL-17 and MMP-9 in the BALF were determined by ELISA. Tregs in the BALF and PB were detected by flow cytometry. Horses with SEA were older than horses with MEA and had higher concentrations of MMP-9 in the BALF (P < 0.05). The neutrophilic inflammation group was characterised by higher age, grade of clinical score, percentage of Tregs and MMP-9 concentration in the BALF compared to the mastocytic inflammation group (P < 0.05). Age positively correlated with neutrophils, grade of score, Tregs in the BALF and in the PB. The grade of score positively correlated with MMP-9. The percentage of Tregs in the BALF positively correlated with Tregs in the PB. Tregs in the BALF and PB positively correlated with neutrophils and MMP-9 in the BALF and with IFN-γ, IL-4 and IL-10 in the plasma. IFN-γ and IL-10 in the plasma positively correlated with the age. The results indicate that the evaluation of selected parameters in PB, the collection of which is less invasive than the collection of BALF, is of potential importance.

Distinguishing between stamping in blood from walking through blood using blood pattern analysis

Bloodstains are typically encountered in violent incidents involving the use of a weapon or physical actions, such as punching, kicking, or stamping. Bloodstain pattern analysis can provide inceptive evidence or intelligence about what happened in an alleged incident, the sequence of events, along with indicating possible suspects if blood is analysed through DNA profiling. This research project focused on the differences in patterns created on footwear during a violent action, such as stamping on a person, and a non-violent action, such as walking through a pool of blood. In this project, several experiments were designed to simulate the stamping and walking actions on a surface wet with blood: carpet, lino flooring, and belly pork meat. Two volunteers with varying body weights were recruited to perform the two actions, using a pair of trainers and a pair of Wellington boots. Defibrinated horse blood was used to simulate real human blood. It was found that the patterns created from stamping and walking through blood differed by the type of pattern and the number and size of stains. The footwear used in the stamping action was characterised by a larger contact stain on the sole than those used in the walking actions: ∼209 mm in length by ∼92 mm in width versus ∼65 mm in length by ∼60 mm in width. The stamping action produced a large number of impact spatters (∼435) on the sides of the footwear versus no impact spatters in the walking actions. The presence of impact spatters was found to be the most prominent feature that differentiated between the two actions. The findings were statistically significant (p < 0.05) and could assist in evaluating whether a defendant was actively involved in a stamping action, or the evidence found was due to innocent reasons.

Behavioral responses of horses (Equus ferus caballus) vary in response to potentially threatening odor condition and aversive social odor stimuli

Horses (Equus ferus caballus) engage their sense of smell in numerous behaviors. However, only few studies so far evaluated responses of horses to olfactory stimuli presumed to be either attractive or aversive and rather focused on presenting horses with only one type of odor. Therefore, we assessed behavioral responses of horses towards seven natural odors that are either potentially threatening, or socially attractive, or socially aversive. To this end, we presented a total of 10 horses with the following odors: same-sex and opposite-sex horse feces; same-sex and opposite-sex horse urine; tiger (Panthera tigris) feces and urine; and horse blood attached to one of two simultaneously presented food buckets and recorded their behavior. We found that the horses displayed clear behavioral reactions towards potentially threatening odors (tiger feces and urine; horse blood) compared to socially attractive (opposite-sex feces and urine) and, in some cases, socially aversive (same-sex feces and urine) odors. More precisely, the horses put their ears back significantly more frequently and tended to do it for longer in the potentially threatening odor condition compared to the socially attractive one. In the potentially threatening odor condition, the horses ate from the control bucket significantly more frequently compared to the other two conditions. Detailed analyses showed that the horses’ behavioral responses were particularly pronounced following exposure to tiger feces and urine. Furthermore, we found that, in some situations, socially aversive odor stimuli caused higher vigilance and more cautious approach, as reflected in increased time of sniffing the odor-bearing bucket and decreased frequency of eating from it in the socially aversive compared to the potentially threatening odor condition. These findings suggest that horses, which are known as prey and social animals, are sensitive to social odors and even more so to odors from predators.

Development of culture methods capable of culturing a wide range of predominant species of intestinal bacteria.

In recent years, with the development of non-cultivation approaches, it has become evident that intestinal bacteria have a significant impact on human health. However, because one-third of the genes cannot be annotated, it is difficult to elucidate the function of all intestinal bacteria by in silico analysis, and it is necessary to study the intestinal bacteria by culturing them. In addition, various media recommended for each individual bacterium have been used for culturing intestinal bacteria; however, the preparation of each medium is complex. To simultaneously culture many bacteria and compare bacterial phenotypes under the same conditions, a medium capable of culturing a wide range of bacteria is needed. In this study, we developed GAM + blood medium (GB medium), which consists of Gifu anaerobic medium containing 5% (v/v) horse blood; it is easy to prepare and it allowed the successful cultivation of 85% of the available predominant species in the human intestinal microbiota.

The effect of halogen-containing anesthetics on the indicators of the gas composition of the blood of horses

The aim of the work was to determine the parameters of the gas composition and acidbase state (CBS) of blood to assess the adequacy of general anesthesia of horses with halogen-containing anesthetics in clinical and instrumental studies. The object of the study was 18 horses aged four to six years, which were castrated. The paper compared the dynamics of blood gas composition indicators at various stages of inhalation anesthesia with fluorotane, isoflurane and sevoflurane in order to prevent and timely detect hypoxemia and hypercapnia in horses. Based on a comprehensive assessment of the clinical examination data, laboratory studies of the gas composition of arterial blood, objectively determine the severity of hypoxemia and hypercapnia, which made it possible to provide the necessary assistance to the animal in a timely manner. The study of blood gas composition indicators has established that the amount of carbon dioxide tension in arterial blood (РaСO2) with both types of anesthesia is subject to the greatest changes during induction and during awakening. During the induction period, it increased both with the inhalation of fluorotane and with the use of isoflurane and sevoflurane, respectively, by 41%, 3.1% and 3.4% compared to the initial values. During the period of awakening, the value of РaСO2 returned to the initial level with anesthesia with isoflurane, with anesthesia with sevoflurane and fluorotane remained above the initial values by 21.8% and 42.8%, respectively. Early detection of signs of hypoxemia and hypercapnia made it possible to avoid the development of further complications during general anesthesia in horses

Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

Cutibacterium acnes has been described as the most common causative microorganism in prosthetic shoulder infections. Conventional anaerobic culture or molecular-based technologies are usually used for this purpose, but little to no concordance between these methodologies (k = 0.333 or less) has been observed. (1) Is the minimum C. acnes load for detection higher for next-generation sequencing (NGS) than for anaerobic conventional culture? (2) What duration of incubation is necessary for anaerobic culture to detect all C. acnes loads? Five C. acnes strains were tested for this study: Four strains were causing infection and were isolated from surgical samples. Meanwhile, the other was a reference strain commonly used as a positive and quality control in microbiology and bioinformatics. To create inoculums with varying degrees of bacterial load, we began with a standard bacterial suspension at 1.5 x 10 8 colony-forming units (CFU)/mL and created six more diluted suspensions (from 1.5 x 10 6 CFU/mL to 1.5 x 10 1 CFU/mL). Briefly, to do so, we transferred 200 µL from the tube with the highest inoculum (for example, 1.5 x 10 6 CFU/mL) to the following dilution tube (1.5 x 10 5 CFU/mL; 1800 µL of diluent + 200 µL of 1.5 x 10 6 CFU/mL). We serially continued the transfers to create all diluted suspensions. Six tubes were prepared per strain. Thirty bacterial suspensions were tested per assay. Then, 100 µL of each diluted suspension was inoculated into brain heart infusion agar with horse blood and taurocholate agar plates. Two plates were used per bacterial suspension in each assay. All plates were incubated at 37°C in an anaerobic chamber and assessed for growth after 3 days of incubation and daily thereafter until positive or Day 14. The remaining volume of each bacterial suspension was sent for NGS analysis to identify bacterial DNA copies. We performed the experimental assays in duplicate. We calculated mean DNA copies and CFUs for each strain, bacterial load, and incubation timepoint assessed. We reported detection by NGS and culture as a qualitative variable based on the identification or absence of DNA copies and CFUs, respectively. In this way, we identified the minimum bacterial load detected by NGS and culture, regardless of incubation time. We performed a qualitative comparison of detection rates between methodologies. Simultaneously, we tracked C. acnes growth on agar plates and determined the minimum incubation time in days required for CFU detection in all strains and loads examined in this study. Growth detection and bacterial CFU counting were performed by three laboratory personnel, with a high intraobserver and interobserver agreement (κ > 0.80). A two-tailed p value below 0.05 was considered statistically significant. Conventional cultures can detect C. acnes at a load of 1.5 x 10 1 CFU/mL, whereas NGS can detect bacteria when the concentration was higher, at 1.5 x 10 2 CFU/mL. This is represented by a lower positive detection proportion (73% [22 of 30]) for NGS than for cultures (100% [30 of 30]); p = 0.004). By 7 days, anaerobic cultures were able to detect all C. acnes loads, even at the lowest concentrations. When NGS is negative and culture is positive for C. acnes , there is likely a low bacterial load. Holding cultures beyond 7 days is likely unnecessary. This is important for treating physicians to decide whether low bacterial loads necessitate aggressive antibiotic treatment or whether they are more likely contaminants. Cultures that are positive beyond 7 days likely represent contamination or bacterial loads even below the dilution used in this study. Physicians may benefit from studies designed to clarify the clinical importance of the low bacteria loads used in this study at which both methodologies' detection differed. Moreover, researchers might explore whether even lower C. acnes loads have a role in true periprosthetic joint infection.

Inflammatory-like status and acute stress response in horses after road transport

This study aimed to evaluate the change of white blood cell count, serum concentration of cortisol, C-reactive protein, albumin and globulin fractions in horse after road transport, and to assess the linkage among hypothalamic–pituitary–adrenal axis (HPA) and inflammatory reaction. From 10 horses blood samples were collected at rest, before road transport (218 km) (BT); after unloaded (AT), 30 and 60 min after unloaded (AT30 and AT60) in order to assess white blood cell count (WBC), serum cortisol, C-reactive protein (CRP), total proteins, albumin, α1-, α-2, β1-, β2- and γ-globulins. WBC, cortisol, CRP, α1-, α-2 and β2-globulins values increased after road transport than rest condition (p < 0.001). Albumin and A/G ratio showed lower values after road transport than rest (p < 0.001). Pearson’s test showed a negative correlation between cortisol and the values of WBC, CRP, α1-, α2-, β1-, β2- globulins, and a positive correlation between WBC and serum concentration of CRP, α1- and α2-, β1-, β2-globulins at AT and AT30. The results showed that road transport evokes an inflammatory like-status in horses. Moreover, the activation of HPA and the onset of acute phase reaction in response to road transport seem to be interconnected with effects on horse’s immune status.

Comparison of The Effectiveness of Campylobacter Growth Ability in Culture Media Added Supplements Lysed Sheep's Blood with Lysed Cow's Blood in Chicken Meat Samples

Blood is a good growth medium for bacteria by preventing the accumulation of toxic oxygen (peroxide and superoxide) and increasing the growth ability of these bacteria. The purpose of this study was to evaluate the ability of lysed sheep blood and lysed cow blood as a substitute for lysed horse blood on Campylobacter growth media, as well as evaluate the growth of Campylobacter from chicken meat samples on culture media supplemented with lysed sheep blood and lysed cow blood compared to the media cultures using lysed horse blood supplements. The concentration of lysed sheep blood, lysed cow blood, and lysed horse blood used was 5% each. The chicken meat samples used in this study were 30 samples. Based on the results, 14 samples of C. jejuni bacteria showed the ability to grow up to 107 and 16 samples up to 106. In the media added with lysed cow blood supplement only one sample was able to grow up to 106, 17 samples grew up to 105, and 12 samples grew up to 104. These results statistically used the one way analysis of variance (ANOVA) test indicating a significant difference significant (P0.05). Further tests using post hoc least significance different (LSD) showed a comparison of the average growth in lysed horse blood, lysed sheep blood, and lysed cow blood significantly different (P 0.05), while the average value of each treatment was the addition of lysed horse blood supplements, lysed sheep blood, and cow blood consecutive lysis was 8.551 ± 0.340, 7.117 ± 0.544, and 5.265 ± 0.548. The addition of lysed sheep blood and lysed cow blood to bolton broth media in this study can be used for C. jejuni isolation. However, the blood of lysed sheep had a higher effectiveness in the ability to grow C. jejuni, which was close to the ability to grow with the addition of lysed horse blood as a control, compared to the addition of lysed cow blood.

Antimicrobial susceptibility testing of Bacteroides species by disk diffusion: The NordicAST Bacteroides study

ObjectivesAntimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp. MethodsA panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole. ResultsA total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80–0.93) for piperacillin-tazobactam, 0.95 (0.91–0.97) for meropenem and 0.89 (0.83–0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of <28 mm in B. fragilis indicated presence of cfiA. Piperacillin-tazobactam had the most false susceptible results. Categorical errors for this antimicrobial were particularly prevalent in cfiA-positive strains, and piperacillin-tazobactam had the highest number of comments describing zone reading difficulties. ConclusionsInter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted.

Influence of autological growth factors on activation of regenerative processes of the superficial digital flexor tendon of horses

Injury of the superficial digital flexor tendon is known for slow and functionally incomplete recovery, which is a problem in the equestrian horse farming. In the clinical studies, we used platelet-rich plasma, obtained from horse blood, and a specially designed scheme of rehabilitation after physical loads for the treatment of injuries of the superficial digital flexor tendon of horses. We analyzed four clinical cases of injuries of the superficial digital flexor of horses, of which 3 cases were treated using platelet-rich plasma, injected under ultrasound-diagnostic control, and one case that was treated without its administration. Ultrasound studies of the superficial digital flexor tendon revealed the following changes: increase in the volume of the tendon, damage to the fiber structure, and absence of fibrosity on the longitudinal images, and hypo-echogenic or anechogenic structure on transverse and longitudinal images. The results of the platelet-rich-plasma treatment of the horses were compared with the control (without utilization of platelet-rich plasma). As seen on the ultrasound images during the intermediate-control stage (2.5–4.0 months after injury), the horses that had received injection of platelet-rich plasma and had undergone the complex of physical exercises were forming new fibers of the superficial digital flexor tendon, which had distinct structure and alignment. This indicated the process of their physiological development. Four and a half months after being injured, the horse that had only received a symptomatic treatment combined with a complex of rehabilitating physical loads had poorly structured fibers in the damaged area, with their disordered alignment, as revealed by the longitudinal images. This suggested the formation of a scar. Transverse images showed the anechogenic areas of the injury suffered by this horse. The method of preparing and administration of platelet-rich plasma is relatively cheap, given the equipment and a qualification of a veterinary doctor. Taking into account its identified efficacy for recovery of the superficial digital flexor tendon, it would be promising to conduct further clinical trials using platelet-rich plasma for treating other injured tendons of horses, which can notably increase the quality of recovered connective tissues.

Assessing the quality of the anaerobic environment — a method developed to support EUCAST disk diffusion of anaerobic bacteria

The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerant Clostridium perfringens strain DSM 25589 (CCUG 75076 and NCTC 14679). The C. perfringens strain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 μg disk. FAA-HB was incubated for 16–20 h at 35–37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25–33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 μg disk and the C. perfringens strain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.

70 Ahiflower (Buglosoides Arvensis) oil supplementation alters whole blood and muscle fatty acid profile in mature horses

The omega-3 (n-3) profile of common plant-based ingredients are derived from α-linolenic acid (ALA). Previous research has indicated that horses lack the ability to convert ALA or other short-chain polyunsaturated fatty acids (SC-PUFA) into the biologically relevant docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Ahiflower (Buglossoides Arvensis) oil is a novel plant-based source of omega-3 fatty acids derived from n-3 stearidonic acid (SDA), and gamma-linolenic acid (GLA), an omega-6 fatty acid (see Table 1 for fatty acid profile). The objective of this trial was to evaluate how supplementation of Ahiflower oil to horses would alter whole blood and muscle fatty acid composition. Mature Quarter Horses (n = 20) were balanced by age, BW, and BCS and assigned to one of 4 treatment groups. LOW (n = 5; BW = 507.12 ± 2.3kg; BCS = 5.4 ± 0.3) was offered 30mL/day of Ahiflower oil. HIGH (n = 5; BW = 517.45 ± 4.5kg; BCS = 5.7 ± 0.1) was offered 60mL/day of Ahiflower oil. FLAX (n = 5; BW = 497.25 ± 3.5kg; BCS = 6.1 ± 0.7) was offered a flaxseed pellet (CP = 15.42%, Total omega-3 = 3.75%) formulated to provide the same amount of total omega-3′s as LOW was offered. PAST (n = 5; BW = 508.14 ± 3.6kg; BCS = 5.2 ± 0.6) was allowed free-choice access to mixed grass pasture (CP = 16.0%, Total omega-3 = 0.79%). All horses were offered 1.81kg/d of Purina® Strategy GX® (CP = 14.24%, Total omega-3 = 0.44%) daily. LOW, HIGH, and FLAX were housed in drylots by treatment group and were offered 2.0% BW as Timothy grass hay (CP = 10.10%, Total omega-3 = 0.35%). On d 0, 30, and 60 blood samples were collected along with a mid-gluteal muscle sample (∼1g). Samples were analyzed for fatty acid profile via LC-HPLC (UF ATCL).Data were analyzed via ANOVA (SAS 9.4). Horses in the LOW group had a 12.5% increase in total omega-3, while horses in HIGH had a 40.1% increase. Similar differences were observed for EPA (LOW = 19.2%; HIGH = 40.1%), and GLA (LOW = 60.1%; HIGH = 167%). Horses in HIGH had a greater increase from baseline in muscle EPA + DHA compared with all other groups (250%). All differences were significant at P < 0.05. Taken together these data indicate that horses had the ability to convert the PUFA's in Ahiflower oil into EPA. Increased EPA in whole blood and muscle tissue potentially allows for increased production of anti-inflammatory eicosanoids and prostaglandins. Further research is necessary to elucidate the mechanism by which this conversion occurs.

Molecular Identification of Culicoides Species and Host Preference Blood Meal in the African Horse Sickness Outbreak-Affected Area in Hua Hin District, Prachuap Khiri Khan Province, Thailand.

African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus Culicoides are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential Culicoides species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, Culicoides were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the Culicoides species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the Culicoides species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female Culicoides were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve Culicoides species identified by morphology were noted, including C. oxystoma (71.92%), C. imicola (20.44%), C. actoni (2.28%), C. flavipunctatus (1.98%), C. asiana (0.99%), C. peregrinus (0.60%), C. huffi (0.60%), C. brevitarsis (0.40%), C. innoxius (0.30%), C. histrio (0.30%), C. minimus (0.10%), and C. geminus (0.10%). The PCR detection of the Culicoides COXI gene confirmed Culicoides species in 23 DNA samples. PCR targeting the PNOC gene revealed that the Culicoides collected in this study fed on Equus caballus (86.25%), Canis lupus familiaris (6.25%), Sus scrofa (3.75%), and Homo sapiens (3.75%) for their blood meal. Human blood was identified from two samples of C. oxystoma and a sample of C. imicola. Three dominant species including C. oxystoma, C. imicola, and C. actoni that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, C. oxystoma, C. imicola, and C. bravatarsis also feed on canine blood. This study revealed the species of Culicoides in Hua Hin district, Thailand, after the AHS outbreak.

Blood from Horses and Cows In Vitro Exposed to Quaternium-15 and Thiacloprid: Haematology and Erythrocyte Osmotic Fragility Alterations

Blood cells are constantly exposed to several pollutants, including xenobiotics, and they can be considered a useful target for pollution exposition of the animal. The aim of this study was to investigate the effects of two xenobiotics (Quaternium-15, a preservative used in personal care products, and Thiacloprid, a neonicotinoid pesticide) on the haematological profile and erythrocyte osmotic fragility (EOF) of equine and bovine blood samples. Ten blood samples from horses and cows were exposed for 24 h to Quaternium-15 at a concentration of 0.1 and 1 mg/L and to Thiacloprid at a concentration of 30 and 60 µg/mL. A decrease in the values of the red blood cells, white blood cells, haematocrit, haemoglobin, mean corpuscular volume, and platelets, and an increase of EOF were found in blood samples exposed to xenobiotics compared to the control. According to the results gathered in the current study, the two xenobiotic compounds herein tested negatively affect the haematological indices causing haemolysis both in cattle and horse blood. This study, despite being preliminary, emphasizes the concept that blood cells are an excellent target for evaluating the effects of xenobiotics.

Limosilactobacillus fermentum KAU0021 Abrogates Mono- and Polymicrobial Biofilms Formed by Candida albicans and Staphylococcus aureus.

Candida albicans and Staphylococcus aureus, representing two different kingdoms, are the most frequently isolated pathogens from invasive infections. Their pathogenic attributes, combined with drug resistance, make them a major threat and a challenge to successful treatments, mainly when involved in polymicrobial biofilm-associated infections. In the present study, we investigated the antimicrobial potential of Lactobacillus metabolite extracts (LMEs) purified from cell-free supernatant of four Lactobacillus strains (KAU007, KAU0010, KAU0021, and Pro-65). Furthermore, LME obtained from the strain KAU0021 (LMEKAU0021), being the most effective, was analyzed for its anti-biofilm property against mono- and polymicrobial biofilms formed by C. albicans and S. aureus. The impact of LMEKAU0021 on membrane integrity in single and mixed culture conditions was also evaluated using propidium iodide. The MIC values recorded for LMEKAU0021 was 406 µg/mL, 203 µg/mL, and 406 µg/mL against planktonic cells of C. albicans SC5314, S. aureus and polymicrobial culture, respectively. The LMEKAU0021 at sub-MIC values potentially abrogates both biofilm formation as well as 24 h mature mono- and polymicrobial biofilms. These results were further validated using different microscopy and viability assays. For insight mechanism, LMEKAU0021 displayed a strong impact on cell membrane integrity of both pathogens in single and mixed conditions. A hemolytic assay using horse blood cells at different concentrations of LMEKAU0021 confirmed the safety of this extract. The results from this study correlate the antimicrobial and anti-biofilm properties of lactobacilli against bacterial and fungal pathogens in different conditions. Further in vitro and in vivo studies determining these effects will support the aim of discovering an alternative strategy for combating serious polymicrobial infections caused by C. albicans and S. aureus.