Horse Blood Research Articles

BIOCHEMICAL PROFILE OF BLOOD IN HORSES DETECTED SUFFERING FROM SURRAIN EAST SUMBA, EAST NUSA TENGGARA, INDONESIA

The infectious animal disease known as surra, or trypanosomiasis in animals, is caused by Trypanosoma evansi.In Indonesia, surra has been recognized as a strategic infectious disease affecting animals. East Nusa Tenggara Province, identified as a center of livestock farming in Indonesia, is also known as an endemic area for surra and requires attention. Therefore, this study was aimed to determine the biochemical profile of blood in horses infected with T. evansi in East Sumba Regency. The parameters observed included blood glucose levels, total protein, creatinine, alanine transaminase (ALT), and aspartate transaminase (AST). Samples were collected from horses raised by the community in East Sumba Regency. The biochemicalexamination was performed using the Photometer 5010V5+ instrument.Blood glucose was examined usingGOD-PAP deproteinization method; totalprotein was determined using a photometry colorimetric test; creatinine was carried using the Jaffe-fixed time kinetic method; ALT and AST were performed using the kinetic method; and UV Test. The results showed that the average biochemical profile of blood in Sumba horses infected with T. evansi was glucose (68.17±5.11mg/dL); protein (5.417±1.82g/dL); creatinine (1.74±0.39mg/dL); urea (28.93±6.26mg/dL); AST (357.00±117.02U/L); and ALT (20.17±6.30U/L). Meanwhile, the average blood biochemical profile in Sumba horses not infected with T. evansi was glucose (99.00±14.96mg/dL); protein (7.20±1.72g/dL); creatinine (1.55±0.51mg/dL);urea (28.13±6.88mg/dL); AST (312.83±94.42U/L); and ALT (23.33±9.45U/L). Blood glucose levels in horsesinfected with T. evansiwere significantly lower (P<0.01) compared to non-infected horses. Apart from blood glucose levels, all observed parameters were in normal ranges.

Relationship between morphometric measurements and blood parameters in horses with varying adiposity levels and physiological conditions.

Understanding and finding the correlation between morphometric measurements and horse blood parameters is crucial for predicting equine metabolic issues. This study aims to analyse morphometric measurements and blood samples in horses with varying adiposity levels. Cross-sectional observational. A total of 50 horses were included in the study and categorized into groups based on their body condition score (BCS) and cresty neck score (CNS). The insulin concentration was significantly higher in overweight horses (p=0.022). Female horses exhibited higher cortisol concentrations (p=0.025) and girth circumference at the withers (p=0.004) compared to males. Lactating mares exhibited higher concentrations of serum total protein (p=0.012) and globulin (p=0.003). A positive correlation was observed between BCS and insulin concentrations (r=0.290, p=0.041). Negative correlations were found between neck circumference to height at withers and glucose (r=-0.309, p=0.029), CNS and glucose (r=-0.315, p=0.026) as well as between crest diameter and cortisol (r=-0.360, p=0.01). Increasing the sample size and conducting longitudinal studies would enhance the study's validity and reliability. Although insulin, glucose and cortisol concentrations have predictive capabilities based on signs and certain morphometric measurements, their correlations are not always strong. Therefore, this study challenges the notion that all overweight horses are unhealthy, as overweight horses can still have good metabolic health. Conversely, lean horses may also experience metabolic issues. Hence, relying solely on visual cues is insufficient to diagnose the metabolic status of horses. Other factors must also be considered to assess their health status accurately.

Phenotypic Shift of an Inflammatory Eosinophil Subset into a Steady-State Resident Phenotype after 2 Years of Vaccination against IL-5 in Equine Insect Bite Hypersensitivity

Eosinophils play a key role in allergic diseases such as insect bite hypersensitivity (IBH). Together with Th2 cells, they shape the course of inflammation in associated type I/IVb allergies. Therefore, a virus-like particle (VLP)-based vaccine targeting equine interleukin-5 (eIL-5), eIL-5-CuMV-TT, was developed to interfere with the IL-5 dependency of eosinophils by inducing the production of anti-self-IL-5 antibodies and alleviating clinical signs in IBH-affected horses. A previous study highlighted the presence of two eosinophil subsets, steady-state resident eosinophils (rEos) and inflammatory eosinophils (iEos), circulating in the blood of healthy and IBH-affected horses, distinguishable by the expression of integrin CD49f. Furthermore, eIL-5-CuMV-TT 1st year vaccination showed a significant decrease of total eosinophils and, in particular, iEos. Nevertheless, the very few remaining eosinophils still shared an iEos phenotype, reflected by bigger size and higher granularity. The aim of this study was to follow up on the phenotype of eosinophils in the 2nd year of vaccination of IBH-affected horses with eIL-5-CuMV-TT. Using flow cytometry analysis of the blood of healthy, IBH, IBH-placebo, and IBH-vaccinated horses, the percentage and count of cells were compared between groups with a focus on pair analysis of eosinophils in 1st and 2nd year vaccinated horses. Our data showed comparably low levels of iEos and a significant increase of rEos in 2nd year compared to 1st year vaccinated horses, suggesting a phenotypic shift toward a resident-like eosinophil population, primarily associated with the phenotype of healthy horses. The reduction of size, granularity, and expression of integrin CD49f in the 2nd year suggests a benefit of long-term treatment with the eIL-5-CuMV-TT vaccine.

Evaluation of the Disk Diffusion Test for Bacteroides fragilis Group Clinical Isolates.

Bacteroides fragilis group (BFG) isolates are the most frequently isolated gram-negative anaerobic bacteria and exhibit higher levels of antimicrobial resistance than other anaerobic bacteria. Reliable susceptibility testing is needed because of reports of resistance to the most active antibiotics. Recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) introduced disk zone diameter breakpoints. We evaluated the disk diffusion test (DDT) for susceptibility testing of BFG isolates compared with the agar dilution method. In total, 150 BFG isolates were collected from three institutes in Korea. The agar dilution method was conducted according to the CLSI guidelines. DDT was performed following the EUCAST guideline. Fastidious anaerobe agar supplemented with 5% defibrinated horse blood was used as the culture medium. Nine antimicrobials were evaluated: penicillin, cefoxitin, cefotetan, imipenem, meropenem, piperacillin-tazobactam, clindamycin, moxifloxacin, and metronidazole. The categorical agreement (CA) between the two methods was >90.0% for imipenem, meropenem, clindamycin, and metronidazole. However, the CA for piperacillintazobactam was low, at 83.2%. Major errors were found: 5.4% for imipenem, 7.4% for meropenem, and 12.8% for piperacillin-tazobactam. All minor errors were <10%. We propose using the area of technical uncertainty (ATU) zone-overlapping area for susceptible and resistant strains to reduce errors in the DDT. Outside the ATU, the CAs of cefoxitin, cefotetan, and piperacillin-tazobactam were >90.0%, whereas that of moxifloxacin was increased to 88.5%. The DDT can be a useful alternative antimicrobial susceptibility test for BFG isolates when using the ATU zone to reduce errors.

Evaluation of gradient diffusion in vitro susceptibility testing of Aerococcus urinae

ObjectiveAerococcus urinae antimicrobial susceptibility testing can be performed via broth microdilution with Mueller-Hinton broth supplemented with lysed horse blood. We sought to compare this with the commonly used gradient diffusion method. MethodsWe compared broth microdilution with Mueller-Hinton broth supplemented with lysed horse blood and gradient diffusion via Mueller-Hinton agar supplemented with sheep blood for 190 A. urinae isolates against 16 antimicrobials. ResultsNo antimicrobials demonstrated more than 90% essential and categorical agreement, and fewer than 3% demonstrated major and very major error rates. Trimethoprim-sulfamethoxazole demonstrated an 81% major error rate and ceftriaxone demonstrated a 76% very major error rate. Agar dilution with lysed horse blood was performed for trimethoprim-sulfamethoxazole against 94 isolates and showed 100% susceptibility, consistent with previous studies. ConclusionsGiven its limitations in detecting resistant strains, our findings cannot support the routine use of gradient diffusion with Mueller-Hinton agar supplemented with sheep blood for A. urinae in lieu of the Clinical and Laboratory Standards Institute method. Our results suggest that A. urinae is usually susceptible to penicillin, linezolid, tetracycline, and vancomycin. Future studies should evaluate alternative testing methods for clinical microbiology laboratories.

Effect of silica-sprayed collection tubes on synovial fluid bacterial culture.

Silica-sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture. The study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no-additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48h. Outcome variables were negative culture, positive culture and total number of colony-forming units (CFUs). Statistical analysis was performed using Mann-Whitney U test, and significance was set at p<0.05. The total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24h. Silica-containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases.

Engineered 3D-printed poly(vinyl alcohol) vascular grafts: Impact of thermal treatment and functionalization

Cardiovascular diseases, a leading cause of global mortality, are driving increased demand for artificial blood vessels for surgical repair. This study discloses the fabrication of three-dimensional (3D)-printed small blood vessels as tissue-engineered grafts. Large-diameter artery and vein grafts are readily available in the market, but small-diameter blood vessels face issues due to the lack of suitable materials. Lysine-biofunctionalized and unmodified poly(vinyl alcohol) grafts (PVA grafts) (2 mm inner diameter and 3 mm outer diameter) suitable for veins and venules were designed using Fusion 360 software, Autodesk Fusion. The PVA channels were fabricated from the 3D virtual model through fused deposition modeling using a PVA filament. These channels underwent thermal treatment to adjust their crystallinity, chemical crosslinking, and functionalization to optimize their mechanical properties and biocompatibility. Crosslinking and biofunctionalization were assessed using Fourier-transform infrared spectroscopy with attenuated total reflectance, while X-ray diffraction and differential scanning calorimetry were utilized for structural analysis. PVA grafts were biologically tested using three specific types of cell cultures: bEnd.3 brain endothelial cells, L929 fibroblast cells, and U937 monocyte-like cells. The hemocompatibility of the optimized vascular grafts was evaluated using horse blood, following the guidelines outlined in ASTM F756-13 Standard Practice for Assessment of Hemolytic Properties of Materials. The direct method for hemoglobin determination was specifically employed. Additionally, we developed an external polyethylene terephthalate glycol (PETG) 3D-printed platform to house the PVA grafts in parallel. The assembled platform (PETG and PVA graft) was connected to both an inlet and an outlet to facilitate the passage of an aqueous flow through the internal section of the PVA grafts during a flow test conducted under simulated body conditions (vacuum and blood pressure: 40 mbar). The flow was induced by a vacuum pump connected to the outlet of the platform, while the inlet was connected to a feeding glass. In summation, we have established a suitable protocol for producing small vascular grafts and demonstrated that the optimization process could significantly affect graft properties.

The effect of solarium light therapy on selected biological and biochemical parameters of peripheral blood in young and old horses.

The aim of the study was to assess the impact of solarium light therapy on selected biological and biochemical parameters of peripheral blood in recreational horses. The study involved 10 horses divided into two groups of young (aged 5 to 7 years) and old (aged 14 to 19 years) individuals. All animals participated in light therapy sessions every other day. Blood was sampled three times during the study: before the treatment, after five light sessions, and after ten light sessions. Morphological parameters, the activity of antioxidant enzymes, TAS values, and the levels of glutathione (GSH), vitamin D3, vitamin C, and malondialdehyde (MDA) were measured in the whole blood. Light therapy contributed to an increase in MCV, HDW, MCVr, CHr and MPV indices, and simultaneously a decrease in the basophil counts, MCHC, RDW and CHCMr indices in both groups of horses (p ≤ 0.05). At the same time reticulocytes fell in older whereas white blood cells and monocytes counts expanded in younger individuals. The treatment also increased the activity of glutathione reductase (GR) and glutathione peroxidase (GPx) in young but decreased the activity of mentioned enzymes in blood plasma of old horses. The total antioxidant status (TAS) of the blood plasma rose progressively, whereas GSH levels declined in all individuals. Moreover, vitamin D3 levels did not change, whereas vitamin C levels gradually decreased during the experiment. The therapy also helped to reduce levels of MDA in the blood plasma, especially of older horses (p ≤ 0.05). In turn, GPx and GR activities as well as MDA levels significantly declined, whereas GSH levels notably elevated in erythrocytes (p ≤ 0.05). Solarium light therapy appears to have a beneficial impact on the morphological parameters and antioxidant status of blood in recreational horses in the winter season. However, the observed results could in part be attributed to the natural physiological adaptation of each individual organism to the treatment.

Nutrient Composition and Feed Hygiene of Alfalfa, Comparison of Feed Intake and Selected Metabolic Parameters in Horses Fed Alfalfa Haylage, Alfalfa Hay or Meadow Hay.

The aim of this study was to examine the nutrient composition and feed hygiene of alfalfa as well as investigate the feed intake, blood, urine and faecal parameters of horses fed alfalfa haylage (AS) compared with alfalfa hay (AH) and meadow hay (MH). A total of 11 geldings were fed ad libitum (2.1% dry matter (DM) of body weight (BW)) with alfalfa haylage, alfalfa hay and meadow hay (MH) in a Latin square design. On days 0 and 21 of the feeding period, blood samples were analysed for kidney and liver parameters. Faecal samples were analysed for pH, DM and short-chain fatty acids (SCFAs). Spontaneous urine was collected during the feeding period to analyse selected parameters. Forage was analysed in terms of feed hygiene and crude nutrients. In several feed samples of AS, AH and MH, the microbial reference ranges were exceeded for product-typical and spoilage-indicating bacteria and fungi. Crude nutrient analyses revealed a median crude protein content of 139 (138/142) g/kg DM for AS, which was similar to that in AH (127-135 g/kg DM) and substantially higher than in MH (79.1-87.7 g/kg DM). The calcium level in AS (11.3 g/kg DM) was significantly higher than that in MH (4.00-4.95 g/kg DM) but not compared with that in AH (9.80-10.4 g/kg DM). All blood parameters were within the reference ranges. Fractional excretion (FE)Ca for AS-fed horses ranged from 8.13 to 22.0%; the FECa for AH-fed horses ranged from 6.48 to 24.8%; the FECa for MH-fed horses ranged from 6.69 to 53.2%. No significant differences were found in faecal pH or SCFA content in AS-fed horses compared with AH-fed and MH-fed horses. We concluded that alfalfa haylage provides an alternative forage for equine nutrition.

Preventing Bacterial Contamination of Breast Implants Using Infection Mitigation Techniques: An In Vitro Study.

Bacterial contamination of implants has been linked to biofilm formation and subsequent infection, capsular contracture, and breast implant-associated anaplastic large cell lymphoma. Reducing contamination during implant insertion should therefore reduce biofilm formation disease sequelae. The aim of this study was to compare levels of contamination between preventative techniques. A model to simulate the passage of implants through a skin incision was designed that utilized a sterile textured polyvinyl plastic sheet contaminated with Staphylococcus epidermidis. In the first stage of the polyvinyl contamination model, implants were subject to infection-mitigation techniques and passed through the incision, then placed onto horse blood agar plates and incubated for 24 hours. In the second stage of the study the same contamination was applied to human abdominal wall specimens. A 5 cm incision was made through skin and fat, then implants were passed through and levels of contamination were measured as described. Smooth implants grew a mean of 95 colony-forming units (CFUs; approximately 1 CFU/cm2) and textured implants grew 86 CFUs (also approximately 1 CFU/cm2). CFU counts were analyzed by the Mann-Whitney U-test which showed no significant difference between implant types (P < .05); independent-sample t-tests showed a significant difference. The dependent-variable techniques were then compared as groups by one-way analysis of variance, which also showed a significant reduction compared with the control group (P < .01). This in vitro study has shown the effectiveness of antiseptic rinse and skin/implant barrier techniques for reducing bacterial contamination of breast implants at the time of insertion.

Effect of clodronate on gene expression in the peripheral blood of horses.

There are two FDA-approved bisphosphonate products, clodronate (Osphos®) and tiludronate (Tildren®), for use in horses. It is hypothesized that bisphosphonates can produce analgesic effects and prevent proper healing of microcracks in bone. Therefore, bisphosphonate use is banned in racehorses. However, bisphosphonates have a short detection window in the blood before sequestration in the skeleton, making the reliability of current drug tests questionable. Seven exercising Thoroughbred horses were administered clodronate (1.8 mg/kg i.m.), and four were administered saline. RNA was isolated from peripheral blood mononuclear cells (PBMCs) collected immediately before a single dose of clodronate or saline and then on Days 1, 6, 28, 56 and 182 post-dose. mRNA was sequenced and analysed for differentially expressed transcripts. While no single transcripts were differentially expressed, pathway analysis revealed that p38 MAPK (p = .04) and Ras (p = .04) pathways were upregulated, and cadherin signalling (p = .02) was downregulated on Day 1. Previously investigated biomarkers, cathepsin K (CTSK) and type 5 acid phosphatase (ACP5), were analysed with RT-qPCR in a targeted gene approach, with no significant difference observed. A significant effect of time on gene expression for ACP5 (p = .03) and CTSK (p < .0001) was observed. Thus, these genes warrant further investigation for detecting clodronate use over time.

Behavioral observations, heart rate and cortisol monitoring in horses following multiple oral administrations of a cannabidiol containing paste (part 2/2).

As a remedy against stress and anxiety, cannabidiol (CBD) products are of increasing interest in veterinary medicine. Limited data is available describing the actual effectiveness of CBD in horses. The aim of this study (part 2 of 2) was to analyze stress parameters via behavioral observation, heart rate monitoring and assessment of blood and saliva cortisol levels in healthy horses treated repeatedly with a CBD containing paste. Twelve horses were randomly assigned to a treatment or a control group. Two pastes were orally administered in a double-blinded study design, one paste containing CBD and one paste without active ingredient. Both pastes were administered twice daily over 15 days (dose: 3 mg CBD/kg). Behavioral observations were conducted daily using a sedation score and a rating of facial expressions, based on the previously described facial sedation scale for horses (FaceSed) and the Horse Grimace Scale. Blood and saliva samples were obtained regularly to determine cortisol levels throughout the study. Cortisol levels were analyzed by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). Behavioral observations and cortisol levels were compared between groups. Prior to paste administration, a novel object test was performed and the horses' reaction to loading on a trailer was recorded. Both tests were repeated after 13 days of paste application. Movement patterns such as different gaits during the novel object test were evaluated and an ethogram was designed to assess exhibited behavioral traits. Cardiac beat-to-beat (R-R) intervals were recorded throughout and evaluated using heart rate (HR) and heart rate variability (HRV) parameters. Blood and saliva samples for cortisol analysis were taken before and after the tests. Daily behavioral observations and cortisol levels did not differ between the treatment and the control group. Similarly, analysis of movement patterns, HR, HRV and cortisol levels during the novel object test and trailer test did not identify significant differences between the groups. Regularly administered oral CBD (3 mg/kg BID over 15 days) had no statistically significant effect on behavioral observations, cortisol levels, HR and HRV in horses. Further research is required to establish adequate doses and indications for the use of CBD in horses.

Leukocyte indexes for equine immune status assessment

Leukocyte indexes, as a diagnostic tool, are highly informative, combined with methodological accessibility. This article provides an overview of various leukocyte indexes. Based on the results of a study of the morphological parameters of the blood of horses, twenty-one leukocyte indexes were calculated and reference values were determined.

Hollow-fibre infection model: adaptations for the culture and assessment of fastidious organisms.

The hollow-fibre infection model (HFIM) is a valuable in vitro platform for emulating antimicrobial drug pharmacokinetic profiles. Despite its potential, standardized protocols for HFIM operation, especially concerning fastidious organisms, are lacking. This study addresses this gap by examining challenges in culturing Pasteurella multocida and Actinobacillus pleuropneumoniae, two fastidious organisms, in the HFIM. Our findings reveal effective strategies to prevent system clogging, involving multiple freeze-thaw cycles of horse blood, centrifugation and cell straining to enhance the clarity of the Mueller-Hinton fastidious medium defined by the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute. Additionally, we propose that the provision of a CO2 atmosphere, along with the utilization of gas-permeable tubing and gas vent filters, significantly facilitates the growth of fastidious organisms. Remarkably, both P. multocida and A. pleuropneumoniae were sustained for a period of up to 10 days under these optimized conditions. This study provides crucial insights into the modifications necessary to successfully culture fastidious organisms in the HFIM, paving the way for more accurate and representative in vitro models for antimicrobial drug testing. These advancements hold promise for advancing research in the field of antimicrobial pharmacokinetics and efficacy against challenging pathogens.

Developing a selective culturing approach for Campylobacter hepaticus.

Campylobacter hepaticus, the causative agent of Spotty Liver Disease (SLD) is an important disease in cage-free egg producing chickens causing mortality and production drops. C. hepaticus is a slow growing Campylobacter easily overgrown by fecal bacteria. It is currently only reliably isolatable from bile samples. A selective media for isolation from feces or environment would assist diagnosis and impact assessment. Growth of five Australian C. hepaticus isolates was studied using Horse blood agar (HBA), sheep blood agar (SBA), Bolton, Preston and Brain Heart Infusion (BHI) base media. Blood and/or bile were added to Bolton, Preston and BHI medias. C. jejuni was used as a positive control. Plates were incubated in duplicate under microaerophilic conditions at 42°C for 10 days and examined at days 3-5 and 7-10 of incubation. Each isolate was examined for sensitivity to 14 antimicrobials using HBA sensitivity plates. Growth was inhibited by BHI and by added bile, while blood improved growth. Further replicates using SBA, HBA, Bolton and Preston media showed best growth on Bolton agar with blood. All five C. hepaticus isolates were resistant to trimethoprim and vancomycin, while four were also resistant to rifampicin and bacitracin. Media based upon Bolton plus blood supplemented with vancomycin and trimethoprim might be used as the most appropriate media for selective growth of C. hepaticus. The addition of bile to media for C. hepaticus isolation and growth will inhibit growth and is not advised.

Prevalence of piperacillin/tazobactam resistance in invasive Haemophilus influenzae in Germany.

Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller-Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations.

Changes in the cellular composition of blood in horses in acute laminitis

Relevance. Equine laminitis is a multi-organ disease that can affect important systems of the horse’s body in the most neglected cases, but most often the hooves are affected, which in turn affects the economic component of the owner of the sick animal.Methods. The work was performed on two groups of horses. The first group included clinically healthy horses, the second group had clinical signs of laminitis. Blood for tests was taken in compliance with a starvation diet and the rules of asepsis and antiseptics from the jugular. The calculation of the leukocyte formula was carried out manually. The results were systematically evaluated and compared with each other to calculate the percentage ratio.Results. It was found that with laminitis there is a significant change in the cellular composition of the blood, in horses of the experimental group there is an increase in leukocytes (by about 302.5%), which may be associated with an inflammatory process in the body that triggers the active production of lymphocytes into the bloodstream. There is also an increase in segmented neutrophils when calculating the leukocyte formula (by 41.52%). This change is most likely caused by traumatic damage to the sole of the hoof. In this case, the increase in segmented neutrophils will be influenced by granulocyte colony stimulating factor (G-CSF), a hormone that stimulates the production and release of neutrophils from the bone marrow.

Comparison of new and old BacT/ALERT aerobic bottles for detection of Candida species.

A new version of aerobic blood culture media has been developed for the BacT/ALERT (bioMérieux) blood culture system. We evaluated the time to detection and yeast cell counts in positive blood cultures for each Candida spp. according to changes in media. Isolates from defibrinated horse blood were inoculated into three types of bottles: the old version of aerobic bottle, new version of aerobic bottle, and anaerobic bottle. All bottles were incubated in the BacT/ALERT Virtuo blood culture system. The time to detection was monitored for each bottle, and yeast cell counts were performed immediately after testing positive, determined via the plate count method. Clinical retrospective data of the candidemia samples before and after aerobic bottle change also were analyzed. The median time to detection was 52.47 hours in the old aerobic bottles versus 19.92 hours in the new aerobic bottles (P < 0.001) for Candida glabrata, and standard and clinical strains showed similar results. C. albicans (27.6 to 24.95 hours) and C. guilliermondii (28.92 to 26.9 hours) had shorter time to detection. However, C. auris (25.43 to 28.25 hours) had a longer time to detection in the new aerobic bottle. The retrospective clinical analysis showed a significant decrease in time to detection (45.0 to 19.4 hours) for C. glabrata, which is consistent with our simulated study result for C. glabrata. As a result of analysis including all blood specimens, C. tropicalis showed a significant delay in time to detection in new aerobic bottles. In an analysis limited to peripheral blood specimens, the time to detection of C. parapsilosis was longer in new aerobic bottles than in old aerobic bottles. Most Candida species did not show remarkable TTD differences, but TTD of C. glabrata was markedly reduced in the New FA Plus bottle. The reduction of time to detection enables faster detection and therapeutic approach for C. glabrata infections.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

BackgroundTheileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype.MethodsA pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically.ResultsEach genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays.ConclusionsThe genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.Graphical abstract

Arsenic Levels and Seasonal Variation in Pasture Soil, Forage and Horse Blood Plasma in Central Punjab, Pakistan.

The present study aimed to determine the accumulation levels of arsenic in pasture soil, forage and animals. An atomic absorption spectrophotometer was used to determine the concentration of metals in the samples of soil, forage and blood plasma of horses. The level of arsenic ranged between 4.26mg/kg (summer) and 4.66mg/kg (winter) in soil samples and 2.67mg/kg (summer) and 2.94mg/kg (winter) in forage samples. In blood plasma samples, the mean arsenic (As) values varied between 1.38 and 1.52mg/L. In the blood plasma samples, the mean As values varied between 1.38 and 1.52mg/L. No statistically significant changes were observed for arsenic concentrations in plasma samples of horses for sampling seasons (p > 0.05). A positive correlation was observed for forage and blood plasma to a certain degree for arsenic but for other media, arsenic correlations were negative and insignificant. It is therefore suggested that regular monitoring of heavy metals in soils/plants/animals is essential to prevent excessive build-up of arsenic.