Streptococcus Zooepidemicus Research Articles

Temperature has a greater effect than salinity on microbial survival in saltwater from a single equine hydrotherapy unit.

To determine the ability of bacteria commonly isolated from equine limb wounds to survive in saltwater obtained from an equine hydrotherapy unit at different salinity concentrations and temperatures. Saltwater samples were obtained over a 2-week period (January 22, 2024 to February 2, 2024) from an equine hydrotherapy unit used for clinical patients, kept at either full salinity per manufacturer recommendations or diluted to half salinity to mimic the dilution that likely occurs in the clinical setting between cases when holding tanks are replenished with tap water only. Samples were then autoclaved to eliminate preexisting bacterial contamination before individual inoculation with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus zooepidemicus. Each inoculated sample was maintained at 2, 22, or 44 °C to represent typical holding tank conditions. The bacterial concentration was determined at each condition every 24 hours up to and including 96 hours. The lower limit of detection was set at 1 CFU/mL. Salinity did not affect bacterial survival. Bacterial concentrations generally decreased with increasing temperature over time. Escherichia coli, S aureus, and S zooepidemicus concentrations decreased to the lower limit of detection at 44 °C by 24 to 48 hours, while P aeruginosa concentrations significantly decreased over 24 hours but remained well above the lower limit of detection. Common bacterial isolates of equine limb wounds can survive in typical saltwater hydrotherapy conditions. Further investigation is warranted to determine the clinical relevance of these findings including protocols for hydrotherapy unit disinfection, wastage of treatment water, and case inclusion/exclusion criteria.

Biochemical characterization of hasB, a UDP-glucose dehydrogenase from the hyaluronic acid high-yield producer Streptococcus zooepidemicus CR003

Hyaluronic acid (HA) is widely used in food and pharmaceutical industries. As a UDP-glucose dehydrogenase, hasB catalyzes the conversion of UDP-Glc to UDP-GlcUA in HA biosynthesis. However, little is known about structural characteristic and enzymatic property of hasB. Owing to high-yield HA production by Streptococcus zooepidemicus CR003, an in-depth analysis of hasB from CR003 was investigated in this work. HasB contains 401 amino acids with a molecular weight of 44.96 kDa, which exhibited 62.69 % homology with that from S. pyogenes. The optimal reaction condition for the purified hasB by heterologous expression was 30 °C and pH 9.0. Cu2+ and Zn2+ significantly inhibited hasB activity, whereas Mg2+ had a stimulatory effect. Km values of hasB for UDP-Glc and NAD+ were 0.1378 and 0.7583 mM, and Vmax were 391.3 and 548.8 μmol/min/mg, respectively. To our knowledge, it is currently the highest reported enzyme catalytic activity of hasB. Molecular docking showed that the binding energies of hasB with UDP-Glc and NAD+ were −731.01 and −314.17 kcal/mol, respectively, and the interaction forces between hasB and the substrate play a crucial role in stabilizing the conformation of protein-substrate complexes. Conclusively, hasB from CR003 showed high catalytic efficiency, which has great application potential for HA production.

An updated description of bacterial pneumonia in adult horses and factors associated with death.

Available descriptive studies on equine pneumonia are outdated or focus on specific horse or bacterial populations. To describe the clinical presentation and bacterial isolates of adult horses with bacterial pneumonia and identify factors associated with death. One hundred sixteen horses >2 years old with bacterial pneumonia. Retrospective case series. Data regarding history, physical examination, clinicopathologic features, treatment, bacterial culture and sensitivity, and outcome were collected and analyzed retrospectively. Historical risk factors were present for 60% of cases, whereas abnormal vital signs on intake were present for <50%. Most horses (58%) underwent at least 1 change of antimicrobial treatment, and 67% received the highest-priority critically important antimicrobials. Streptococcus zooepidemicus was the most isolated bacteria (44%), followed by Escherichia coli (19%), Klebsiella spp. (18%), other Streptococcus species (17%), and Bacillus spp. (13%). Fusobacterium spp. were the most common anaerobic isolates (11%). Antimicrobial susceptibility varied widely. Survival to discharge was 73%. Heart rate at presentation (odds ratio [OR] 1.08, 95% confidence interval [CI] 1.008-1.17, P = .03) and higher creatinine (OR 14.1, 95% CI 1.56-127.6, P = .02) increased the risk of death. Higher lymphocyte count (OR 0.27, 95% CI 0.08-0.94, P = .04) reduced risk. Contrasting older literature, Fusobacterium spp. were the most common anaerobes. Streptococcus zooepidemicus remained the most common isolate and was predictably susceptible to penicillin. Antimicrobial susceptibility was otherwise variable and broad applicability is limited as this was a single-center study. Increased risk of death associated with tachycardia and abnormally high serum creatinine concentration is consistent with previous studies.

Separation and purification of hyaluronic acid by [formula omitted] nano and micro particles coated with chitosan and silica

Hyaluronic acid (HA), a glycosaminoglycan, is comprised of alternating units of D-glucuronic acid and N-acetylglucosamine. This compound harbors numerous biomedical applications, including its use in pharmaceuticals, wound healing, osteoarthritis treatment, and drug delivery. Its unique composition and exceptional features, such as its high water-absorbing and retaining capacity, have also led to its use in the cosmetics industry. The employment of this biopolymer has given rise to an escalation in the request for its manufacture. The present investigation has explored the correlation between hyaluronic acid and chitosan and silica for the purpose of separation. Consequently, Iron oxide magnetic nano particles and micro particles were produced via co-precipitation method and were layered with chitosan and silica to purify the hyaluronic acid from the fermentation broth that was generated by Streptococcus Zooepidemicus. The size distribution and zeta potentials of the two kinds of particles were gauged with the aid of a dynamic laser light scattering apparatus and zeta potential meter (Malvern, Zeta master) respectively. The confirmation of the chemical structure of the Fe3O4 nanoparticles and Fe3O4 particles conjugated with chitosan and silica was accomplished through the utilization of Fourier Transform Infrared Spectroscopy (FT-IR). Protein contamination was thoroughly characterized by means of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Nanodrop 2000/2000c spectrophotometers protein estimation method. The maximum HA adsorption capacity, under optimal pH conditions of 4, was determined to be 87 mg/g, 112 mg/g, 51 mg/g, and 44 mg/g for Fe3O4 −chitosan nanoparticle, Fe3O4 −chitosan micro particle, Fe3O4 −silica microparticle, and Fe3O4 −silica nanoparticle, respectively.

Animal Shelter Wastewater Sampling for Pathogens With Epidemic Potential

Canine distemper virus (CDV), canine parvovirus (CPV), and Streptococcus zooepidemicus (S. zoo) represent a range of infectious agents that can cause epidemic disease in dogs, particularly those housed in animal shelters. Outbreaks of disease associated with these agents are relatively common in shelters, resulting in isolation, quarantine, euthanasia, and in some cases, shelter closure in efforts to manage the outbreak. In recent years, outbreaks of CDV and S. zoo have been particularly challenging for shelters in Arizona. Wastewater-based epidemiology (WBE) has proven useful in disease monitoring and outbreak prediction in human populations, including congregate locations, and we hypothesized that it may also be useful in surveillance and prediction for agents responsible for either gastrointestinal (GI) or respiratory disease in shelter animals. Our specific aims were to develop and validate a protocol for detecting infectious agents from wastewater samples collected from shelter run sections and to investigate associations with agent burden and counts of animals with clinical signs. Once protocols for sampling and preparation were optimized, environmental wastewater samples were collected weekly between November 2023 and April 2024 at a large open intake municipal animal shelter in Phoenix, Arizona. The samples were collected from seven drains located in seven run sections in three separate buildings, and were evaluated for the presence of the three agent nucleic acids using quantitative, multiplex PCR. The counts of dogs actively showing GI and respiratory symptoms, graded as mild, moderate, and severe, were collected the afternoon of the day of sampling. Biosafety committee approval was granted for the viruses in this study. Shedding of CPV was found to be widespread and variable over time. S. zoo was detected five times on five different weeks in five different run sections. CDV was found once. There were only six dogs reported with GI signs during the study period. Counts of respiratory disease ranged from 42-132 dogs per run section with most cases being mild in severity and reported in the isolation run section. There is no apparent association between counts of either respiratory or GI cases and the three agents detected through wastewater sampling. During the study there was a single incident of a S. zoo PCR positive dog being isolated and later dying at the shelter. S. zoo was detected through wastewater sampling the week prior to the incident, however, in a run section located in a different building than where the PCR positive dog was housed. To the authors’ knowledge, this study is the first of its kind to develop protocols to detect critical pathogens in wastewater samples from an animal shelter. CPV has been the pathogen most frequently detected, which we speculate may be largely due to the shedding typically observed in dogs post-vaccination, given the absence of GI signs. Further investigation is warranted to better characterize the absence of amplifiable CDV nucleic acids and to examine these samples for other common shelter pathogens.

Whole-Genome Sequencing and Phenotypic Analysis of Streptococcus equi subsp. zooepidemicus Sequence Type 147 Isolated from China.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the important zoonotic and opportunistic pathogens. In recent years, there has been growing evidence that supports the potential role of S. zooepidemicus in severe diseases in horses and other animals, including humans. Furthermore, the clinical isolation and drug resistance rates of S. zooepidemicus have been increasing yearly, leading to interest in its in-depth genomic analysis. In order to deepen the understanding of the S. zooepidemicus characteristics and genomic features, we investigated the genomic islands, mobile genetic elements, virulence and resistance genes, and phenotype of S. zooepidemicus strain ZHZ 211 (ST147), isolated from an equine farm in China. We obtained a 2.18 Mb, high-quality chromosome and found eight genomic islands. According to a comparative genomic investigation with other reference strains, ZHZ 211 has more virulence factors, like an iron uptake system, adherence, exoenzymes, and antiphagocytosis. More interestingly, ZHZ 211 has acquired a mobile genetic element (MGE), prophage Ph01, which was found to be in the chromosome of this strain and included two hyaluronidase (hyl) genes, important virulence factors of the strain. Moreover, two transposons and two virulence (virD4) genes were found to be located in the same genome island of ZHZ 211. In vitro phenotypic results showed that ZHZ 211 grows faster and is resistant to clarithromycin, enrofloxacin, and sulfonamides. The higher biofilm-forming capabilities of ZHZ 211 may provide a competitive advantage for survival in its niche. The results expand our understanding of the genomic, pathogenicity, and resistance characterization of Streptococcus zooepidemicus and facilitate further exploration of its molecular pathogenic mechanism.

Microbial hyaluronic acid production in the 21 century: a roadmap toward high production, tailored molecular weight

Hyaluronic acid is a biopolymer that has gained attention from both researchers and the public since its discovery in 1934. In 2022, its global annual demand was 667 MT, with a market value of 9.4 billion dollars and an expected compound growth rate of 7.7% from 2024 and 2030. Such popularity lies in its many applications, which ranges from pharmaceutical to Tissue Engineering. Its molecular weight (MW) determines which application hyaluronic acid is better suited for: high MW (over 1 MDa) is used in medical applications such as osteoarthritis and ophthalmology, and low MW (less than 0.05 MDa) are suited for moisturizing cosmeceuticals. This makes hyaluronic acid molecular weight an important parameter for its production. The first industrial process was based on extraction from rooster combs. However, it shifted toward microbial production with Streptococcus zooepidemicus. Studies focus on improving its production and tailoring its molecular weight by accessing optimum temperature, pH, aeration, agitation, carbon, and nitrogen sources concentration and origin. his review aims to provide an overview of the current knowledge on the effects of metabolic and environmental factors in microbial hyaluronic acid production. The review shows that there has been extensive research in optimizing cultivation medium composition, temperature, pH, and aeration between 2018 and 2023, but further studies are required to optimize the metabolism of S. zooepidemicus towards maximum biopolymer production and molecular weight.

Streptococcus zooepidemicus in dogs: Exploring a canine pathogen through multilocus sequence typing

Streptococcus equi. subsp. zooepidemicus (S. zooepidemicus) associated diseases in dogs have emerged as a significant concern over recent decades. S. zooepidemicus occurs sporadically in dog populations globally, with increased prevalence in shelters/kennels. This study used multilocus sequence typing (MLST) of 149 independent canine S. zooepidemicus isolates to assess associations between sequence type and breed, country of origin, disease severity, sampling type, year, and behaviour within an outbreak. No clear associations for breed, country, sampling type and year were determined in this study. ST-10 and 123 strains were present within all disease categories, from no clinical signs to severe disease. Assessment of S. zooepidemicus infection in 3 UK outbreaks at the same location found ST-10, 18, 123 strains, and a ST-173 strain in a US outbreak, were associated with haemorrhagic pneumonia and persisted in kennelled populations over time. The ST-173 clonal complex has been noted to have severe virulence capabilities in dogs and other species. S. zooepidemicus seems to thrive in environments with a high risk of transmissibility, overcrowding, stress and naïve populations, particularly for those in shelters/kennels. MLST alone cannot determine the virulence phenotype of S. zooepidemicus in dogs. However, a level of conservancy and diversity within ST allelic loci aids the opportunity to cause severe disease in dogs. Thus, further research into whole genome sequencing and characterising the virulence factors of S. zooepidemicus is warranted in dogs.

Lack of Glutamate Synthase in Streptococcus zooepidemicus makes a Natural Driving Force for Increasing Hyaluronic Acid Production

Lack of Glutamate Synthase in Streptococcus zooepidemicus makes a Natural Driving Force for Increasing Hyaluronic Acid Production

Comparison of In Vitro Bacterial Susceptibility to Common and Novel Equine Wound Care Dressings.

Antimicrobial resistance is becoming a problem of concern in the veterinary field, necessitating the use of effective topical treatments to aid the healing of wounds. Honey has been used for thousands of years for its medicinal properties, but in recent years medical-grade Manuka honey has been used to treat infected wounds. The goal of this study was to determine the relative susceptibility of four common equine wound pathogens to ten different types of antimicrobial agents based on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The pathogens studied include ATCC lab-acclimated Pseudomonas aeruginosa, Escherichia coli, and methicillin-resistant Staphylococcus aureus and one from an equine sample submitted to the Colorado State Veterinary Diagnostic Laboratory (Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus)). An additional goal of the study was to describe the comparison of bactericidal activity of medical-grade Manuka honey, local honey, and commercial, food-grade honey to other commonly used wound dressings (20% hypertonic saline, silver sulfadiazine cream, PHMB gauze, and PHMB foam). The objective is to provide veterinary practitioners with comparative data on the use of a variety of antimicrobial dressings for inhibiting the growth of common wound bacteria. MIC and MBC for Manuka, store, and local honeys were comparable to those of sterile gauze, sugar, and hypertonic saline. Across bacterial species, local honey proved to have more bactericidal activity when compared to Manuka honey and commercial, food-grade honey. The MIC and MBC for PHMB gauze and foam was consistently at a higher dilution compared to the other antimicrobials. The majority of antimicrobials exhibited stronger inhibitory and bactericidal activity against a Streptococcus zooepidemicus isolate obtained from a wound compared to other bacteria that were ATCC lab-acclimated. Additional research for in vivo applications needs to be done to see whether differences exist in effective wound management.

Influence of initial glucose and lactic acid concentration on hyaluronic acid production by Streptococcus zooepidemicus: Kinetic and modeling

Hyaluronic acid (HA) is a molecule of significant commercial value with applications in the pharmaceutical, cosmetic, and medical industries. It is typically produced through microbial fermentation. However, the efficiency of this type of process can be affected by inhibition phenomena due to the substrate or the product. In this sense, the effect of different initial concentrations of glucose (9.3–59.9 g/L) and lactic acid (10.7–56.0 g/L) was studied in the present investigation to determine their effect on the growth of Streptococcus zooepidemicus and HA production in batch fermentation. The maximum HA concentration obtained was 3.3 g/L from 59.9 g/L glucose, while productivity, yield, and specific rate of HA formation were higher at 31.9 g/L glucose. The biomass produced followed a stepwise pattern with increasing glucose, although the specific growth rate decreased after reaching 39.1 g/L glucose. The initial presence of lactic acid (LA) hurt fermentation, decreasing the amount of biomass and HA produced; the inhibitory effect of LA was more pronounced at 19.4 g/L and above, decreasing productivities, yields, and specific rates. Compared to experimental data, the proposed mathematical model described satisfactorily (R2 > 0.9) the dynamics of the fermentation process at different culture conditions.

DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids.

Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent Ct vs log10 (DNA amount) standard curves, which have a linearfit with R2 value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). We show that our strategy is able to successfully detect DNA at as low as 800 copies/μL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.

Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Downstream process intensification for biotechnologically generated hyaluronic acid: Purification and characterization

Hyaluronic acid (HA), an anionic, non-sulfated glycosaminoglycan, has several clinical applications. This study examines several downstream methods for purifying HA with maximum recovery and purity. Following the fermentation of Streptococcus zooepidemicus MTCC 3523 to produce HA, the broth was thoroughly purified to separate cell debris and insoluble impurities using a filtration procedure and a variety of adsorbents for soluble impurities. Nucleic acids, proteins with high molecular weight, were successfully removed from the broth using activated carbons and XAD-7 resins. In contrast, insoluble and low molecular weight impurities were removed using diafiltration, with HA recovery of 79.16% and purity close to 90%. Different analytical and characterization procedures such as Fourier transform-infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy validated the presence, purity, and structure of HA. Microbial HA showed activity in tests for 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging (4.87 ± 0.45kmolTE/g), total antioxidant capacity (13.32 ± 0.52%), hydroxyl radical-scavenging (32.03 ± 0.12%), and reducing power (24.85 ± 0.45%). The outcomes showed that the precipitation, adsorption, and diafiltration processes are suitable for extracting HA from a fermented broth under the chosen operating conditions. The HA produced was of pharmaceutical grade for non-injectable applications.

Identification of endometrial biofilm-specific proteins for in vivo diagnostics

Bacteria enter the uterus in a free-floating planktonic state, which makes them vulnerable to the host-immune system as well as exposure to effective antimicrobials. If not eliminated in a timely fashion, the bacteria will adhere to the endometrial surface and may form micro-colonies, develop a complex matrix, and switch into a biofilm community. The ability of bacteria to shift from planktonic to biofilm phenotypes has been described for both Gram-negative and Gram-positive bacteria in human mucosal tissue, indwelling catheters, and implants as well as other organs, including the equine endometrium. Unfortunately, there is currently no available assay to diagnose the condition in vivo. Literature in human medicine suggests that biofilm matrices contain both secretory and non-secretory proteins, and while some of the proteins are common among all biofilms, others may be specific for bacterial species. Therefore, the objective of this research was to identify specific proteins from biofilm-producing bacteria from mares with endometritis. Whole-cell extracts from potentially biofilm-forming bacterial isolates of Streptococcus zooepidemicus (SEZ) (n=3), Pseudomonas aeruginosa (n=1), and Escherichia coli (n=4) were analyzed by 2-D PAGE. Differentially expressed proteins were selected by overlapping 2-D PAGE biofilm proteins with planktonic culture proteins.In-gel analysis of differences (with a 3-fold cut-off)identified a total of 96 proteins for SEZ, 83 proteins for P. aeruginosa, and 69 proteins for E. coli as potential biofilm markers. Select proteins were further identified by MALDI-TOF/TOF. In SEZ, glutathione-disulfide reductase (48.8 kDa, PI 5.6), which catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant, and TIGR00266 (25.3 kDa, PI 5.4) were identified as proteins of interest. For P. aeruginosa, thioredoxin A (11.8 kDa, PI 4.7), which participates in various redox reactions, osmotically inducible protein C (15.6 kDa, PI 5.9), important for cellular defense mechanism against oxidative stress, and bacterial extracellular solute-binding s, 3 family protein (33 kDa, PI 8.3) serve as potential targets. Potential biomarker candidates for E. coli include the serine protease pet autotransporter (139 kDa, PI 6.5) and outer membrane protein X (18 kDa, PI 6.6), a protein involved in numerous physiological processes including invasion, signal transduction, and binding external proteins. In conclusion, we identified biofilm-specific proteins that may serve as in vivo diagnostic markers for biofilms in uterine lavage from mares with biofilm-associated bacterial endometritis. Future research will pursue these proteins of interest for their use as diagnostic tools in the field.

Bacterial development in semen stored at ambient temperature in INRA96®

Sperm motility and mitochondrial activity can be maintained at room temperature for 24h in INRA96®. However bacterial development remains possible. Furthermore, very few studies evaluated the sensitivity of bacteria to the antibiotics included in INRA96. In experiment 1, semen from 8 stallions (28 ejaculates) was diluted to 20 × 106 sperm/mL in INRA96. The total amount of bacteria, dead or alive, was evaluated using Easykit6® (IMV-Technologies) immediately after dilution, then after 24h and 48h. Bacterial concentration remained stable during 48h cooled storage in Neopor® box (Minitube): mean±sem 9.8±2 × 105, 8.5±2 × 105, 8.4±12 × 105 bacteria/mL at 0h, 24h, 48h, respectively. Bacterial concentration significantly increased after 48h at ambient temperature between 13 and 26°C: 9.7±2 × 105a, 10±2 × 105ab, 28±6 × 105b bacteria/mL at 0h, 24h, 48h, and was significantly higher at ambient temperature than in cooled samples both after 24h and 48h. However, when data were analysed individually, the significant increase occured only for 3 of the 8 stallions. In experiment 2, semen from 7 stallions with 3 ejaculates each was evaluated in duplicate after 0h, 24h and 48h at ambient temperature. Easykit6® showed a significant increase in bacterial concentration for 2 stallions. Conventional bacteriology demonstrated an increase in CFU concentration for 3 stallions including the 2 mentioned above, and a decrease for another stallion. For example, stallion S showed 8±8a, 592±298ab, 8567±4493bCFU/mL at 0h, 24h, 48h, respectively, whereas stallion L showed 1259±504b, 92±53a,117±72aCFU/mL. Sixteen bacterialspecies (38 strains) and one yeast were identified. All the strains of Enterococcus fecalis, Listeria grayi, Streptococcus zooepidemicus, and some strains of Aerococcus viridans, Aeromonas salmonicida, Pasteurella multocida, Pseudomonas sepacia, Staphylococcus hominis, Staphylococcus xylosus, were resistant to both antibiotics of INRA96: 0.028g/L penicillinG and 0.077g/L gentamicin. Ten of the 20 strains identified at 0h and resistant to those antibiotics were not observed anymore at 24h. Eighteen of the 20 strains identified at 48h were resistant to those antibiotics. Eight of them have not been detected at 0h. Candida famata and the 17 bacterial strains identified at 0h and sensitive to those antibiotics were not observed anymore at 24h.The antibiotics and amphotericin B present in INRA96 seem enough to prevent microbial development at ambient temperature, provided that micro-organisms are sensitive to those molecules. Competition occurs between strains: those sensitive to the antibiotics of the extender cannot develop during storage, whereas others, even under the level of detection in raw semen, grow during storage because they are resistant. Bacterial concentration was not significantly correlated with sperm quality (motility, morphological abnormalities). Such bacterial level may not affect spermatozoa. Whether it could induce endometritis in inseminated mares needs investigation. Financial support: IFCE scientific council (Spermicrobes).

Comparison of serum amyloid A concentrations in mares with experimentally induced placentitis to normal, pregnant mares using a commercially available, stall-side device

Ascending bacterial placentitis, a significant cause of pregnancy loss in mares, is challenging to diagnose. Diagnostic tools for ascending placentitis include detection of premature mammary development, vulvar discharge, and ultrasonographic changes to the caudal fetal membranes. These tools have limited sensitivity. Changes in serum biomarkers (ie, serum amyloid A, SAA) provide useful, adjunct information for diagnosing placentitis, but point-of-care assays are limited. The goal of this study was to evaluate a quantitative, stall-side test (Stablelab®, Zoetis, NJ, USA) to measure SAA concentrations in plasma from mares with experimentally induced placentitis compared to normal pregnant mares. We hypothesized that SAA concentrations in mares with placentitis would be higher than normal pregnant mares and that changes in SAA would be predictive of placentitis. Placentitis was induced in 12 mares (270-300 days gestation) with trans-cervical deposition of 1 × 107 Streptococcus zooepidemicus directly onto the chorioallantois. Infected mares were randomly assigned to: infected/untreated (UNTREATED; n=6); or infected/firocoxib (FIRO; n=6; administered firocoxib two days prior to infection until abortion/delivery). Gestationally age-matched, normal pregnant mares were assigned to NORM (n=6). Plasma samples were obtained from mares prior to the study (Day 0) and daily until abortion/delivery. Mares from NORM were sampled similarly to infected, gestation-matched mares. Duplicate plasma samples from post-infection Days 0-9, and peripartum, were evaluated for SAA concentrations using a Stablelab® system, permanufacturer's instructions. SAA concentrations from mares were compared between groups (UNTREATED, FIRO, NORM). To normalize distribution, data were log-transformed and groups compared (geometric means ± SE) using a two-sided t-test. Differences were not identified between infected mares (UNTREATED/FIRO), so SAA concentrations from all infected mares were grouped (INFECT; n=12) and compared to NORM (n=6). Significance was set at P < 0.05. SAA concentrations were significantly higher in INFECT than NORM by Day 4 post-infection (17.81µg/mL± 10.44 v. 0.76µg/mL± 0.62) and remained significantly higher through Day 9 post-infection (412.32µg/mL± 306.67 v. 0.40µg/mL± 0.04). Prior to foaling, SAA concentrations for INFECT were significantly greater than NORM at -48h (34.72 µg/mL± 23.07 v. 0.41µg/mL± 0.31); -24h (85.32 µg/mL± 48.09 v. 0.6µg/mL± 0.58); at foaling (130.19 µg/mL± 68.97 v. 0.41µg/mL± 0.31); and 24h after foaling (468.25 µg/mL± 224.15 v. 7.63 µg/mL± 4.95). These data showed that mares with experimentally induced placentitis had higher concentrations of SAA than normal foaling mares, early after infection and prior to foaling. Differences in SAA concentrations were detectable using a commercially available, stall-side product (Stablelab®, Zoetis). Monitoring SAA, with a point-of-care assay, may provide useful, adjunct information for diagnosing bacterial placentitis in mares. Funding: Grayson Jockey Club; Zoetis; Merial; Kentucky Thoroughbred Breeders, University of Florida

Effects of firocoxib, trimethoprim sulfamethoxazole and altrenogest on inflammation and foal survival after administration to mares with experimentally induced placentitis

Bacterial placentitis is a leading cause of pregnancy loss in mares. Infection, inflammation and prostaglandin production induce preterm delivery leading to poor foal survival. Treatment strategies for placentitis that control infection and inhibit inflammation are vital. The goals of this study were: 1. Assess the anti-inflammatory effects of firocoxib, trimethoprim sulfamethoxazole (TMS) and altrenogest administered to mares with placentitis; 2. Determine the efficacy of firocoxib, TMS and altrenogest for improving foal outcome in mares with placentitis. We hypothesized that this treatment would significantly decrease pro-inflammatory mediators in fetal fluids and tissues when compared to untreated, infected mares and would also improve foal survival. Placentitis was induced in 13 pregnant mares (270-300 days gestation; via intracervical inoculum of Streptococcus zooepidemicus). Mares were randomly assigned to: infected/untreated (UNTREATED; n=6); or infected/treated groups (TREATED; n=7; administered firocoxib, TMS, and altrenogest from the onset of clinical signs of placentitis until abortion/delivery). Fetal fluids, membranes and tissue samples were obtained at abortion/delivery for quantification of TNF-α, IL-1β, IL-10, IL-6, IL-8, PGE2, PGF2α, and matrix metalloproteinases (MMP) -1, -3, -9 using ELISA (fluids) and real-time PCR (tissues). P < 0.05 was considered significant. Gestation length and foal survival were significantly higher in TREATED (mean days post-infection = 40; 7/7 live foals) than UNTREATED (mean days post-infection = 14; 2/6 live foals). Administration of firocoxib, TMS andaltrenogest resulted in significantly lower amniotic fluid concentrations of IL-1β, IL-6, PGE2 and PGF2α in TREATED than UNTREATED. Amniotic fluid IL-10 concentration tended (P < 0.1) to be lower in TREATED than UNTREATED. Cytokine and prostaglandin concentrations in allantoic fluid were not different between groups except PGE2 which tended (P < 0.1) to be lower in TREATED than UNTREATED. The relative quantity of cytokine and MMP mRNA expression in fetal membranes and foal tissues (non-survivors) was log-transformed and estimated marginal means determined. Fetal membranes from TREATED had significantly lower mRNA expression for IL-8 and TNFα compared to membranes from UNTREATED. mRNA expression of IL-1β, IL-6 and MMP3 tended (P < 0.1) to be lower in membranes from TREATED than UNTREATED. mRNA expression of IL-1β, IL-8, IL-10, TNFα, MMP1, 3 and 9 were significantly different between fetal membrane sites. In non-surviving foals, IL-8 mRNA expression was significantly lower in liver than lung while MMP3 expression was significantly higher in liver than lung or spleen. These data provide physiologic evidence that administration of firocoxib, TMS and altrenogest to mares with placentitis has broad anti-inflammatory effects and may improve foal survival. Research funding: Grayson Jockey Club Research Foundation; Merial Animal Health; Kentucky Thoroughbred Breeders Association, University of Florida, University of Georgia.

Parasitological and Bacteriological Evaluation of Common Raw Fruits Collected from Two Major Markets in Iwo, Osun State, South-West Nigeria.

Fruits are a vital part of human nutrition because they contain the growth factors required to maintain normal health. Fruits are also known to harbour a wide range of parasites and bacteria. Consumption of unwashed raw fruits can lead to foodborne pathogens. This study was carried out to examine the presence of parasites and bacteria on fruits sold at two major markets in Iwo, Osun state, South-West Nigeria. Twelve different fresh fruits and seven different fresh fruits were purchased from different vendors from "Odo-ori" and "Adeeke" markets, respectively. The samples were transported to the microbiology laboratory of Bowen University, Iwo, Osun state for bacteriological and parasitological analysis. The parasites were concentrated by sedimentation and were examined using a light microscope, while for the microbial analysis, culturing and biochemical tests were also carried out on all the samples. The parasites discovered include Fasciola hepatica eggs, Ascaris lumbricoides and Strongyloides stercoralis larvae, hookworm larvae, and Taenia and Trichuris trichiura eggs. Ascaris lumbricoides was most frequently detected (40.0%). Bacteria isolated from the sampled fruits include Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli, Kluyvera ascorbata, Proteus mirabilis, Staphylococcus intermedius, Staphylococcus sp., Micrococcus sedentarius, Corynebacterium diphtheriae, and Streptococcus zooepidemicus. The presence of parasites and bacteria on the fruits observed indicates that public health diseases may arise from their consumption. Awareness and education among farmers, vendors, and consumers about the importance of personal and food hygiene through proper washing or disinfection of fruits could reduce the risk of parasites and bacterial fruit contamination.

Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3–L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase SzHYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L SzHYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides.