Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

Intranasal booster vaccination of beef steers reduces clinical signs following experimental coinfection with BRSV and BHV-1 without reducing shedding of BRD-associated bacteria.

OBJECTIVE: To determine the efficacy of primary or booster intranasal vaccination of beef steers on clinical protection and pathogen detection following simultaneous challenge with bovine respiratory syncytial virus and bovine herpes virus 1. METHODS: 30 beef steers were randomly allocated to 3 different treatment groups starting at 2 months of age. Group A (n = 10) was administered a single dose of a parenteral modified-live vaccine and was moved to a separate pasture. Groups B (n = 10) and C (10) remained unvaccinated. At 6 months of age, all steers were weaned and transported. Subsequently, groups A and B received a single dose of an intranasal modified-live vaccine vaccine while group C remained unvaccinated. Group C was housed separately until challenge. Two days following vaccination, all steers were challenged with bovine respiratory syncytial virus and bovine herpes virus 1 and housed in a single pen. Clinical and antibody response outcomes and the presence of nasal pathogens were evaluated. RESULTS: The odds of clinical disease were lower in group A compared with group C on day 7 postchallenge; however, antibody responses and pathogen detection were not significantly different between groups before and following viral challenge. All calves remained negative for Histophilus somni and Mycoplasma bovis; however, significantly greater loads of Mannheimia haemolytica and Pasteurella multocida were detected on day 7 postchallenge compared with day -2 prechallenge. CLINICAL RELEVANCE: Intranasal booster vaccination of beef steers at 6 months of age reduced clinical disease early after viral challenge. Weaning, transport, and viral infection promoted increased detection rates of M haemolytica and P multocida regardless of vaccination status.

NMR-based metabolomics of plasma from dairy calves infected with two primary causal agents of bovine respiratory disease (BRD).

Each year, bovine respiratory disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling for early diagnosis represents a promising tool for developing effective measures for disease management. Here, 1H-nuclear magnetic resonance (1H-NMR) spectra were used to characterize metabolites from blood plasma collected from male dairy calves (n = 10) intentionally infected with two of the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 46 metabolites (BRSV = 32, MH = 33) changed in concentration compared to the uninfected state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Furthermore, 1H-NMR spectra from samples from the uninfected and infected stages were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development, understanding, and validation of novel diagnostic tools.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

The titers, duration, and residual clinical protection of passively transferred nasal and serum antibodies are similar among beef calves that nursed colostrum from vaccinated or unvaccinated dams and were challenged experimentally with bovine respiratory syncytial virus at three months of age.

OBJECTIVES: To compare initial titers, duration, and residual clinical protection of passively transferred bovine respiratory syncytial virus (BRSV) nasal immunoglobulin (Ig) G-1 and IgA, and serum neutralizing (SN) antibodies. ANIMALS: 40 three-month-old beef steers born either to unvaccinated or vaccinated cows. PROCEDURES: During the last trimester of gestation, cows were assigned randomly to either vaccinated or unvaccinated groups. Calves were grouped on the basis of whether they nursed colostrum from unvaccinated dams (NO-VACC group; n = 20) versus dams vaccinated with 2 doses of an inactivated BRSV vaccine (VACC group; n = 20). At 3 months of age, calves were challenged with BRSV. Respiratory signs were scored. Nasal BRSV IgG-1 and IgA and SN antibodies were compared before and after the challenge. The presence of BRSV in nasal secretions was evaluated by reverse transcription-PCR assays. RESULTS: Respiratory scores after BRSV challenge were similar between treatment groups. Nasal BRSV IgG-1 and SN antibodies were significantly greater in VACC calves at 48 hours of life; however, by 3 months of age, titers had decayed in both groups. Nasal BRSV IgA titers were minimal after colostrum intake and before the BRSV challenge, and increased in both groups after the challenge. The NO-VACC group had a significantly greater probability of shedding BRSV compared with VACC calves. CLINICAL RELEVANCE: At 3 months of age, titers of passively transferred BRSV antibodies in VACC and NO-VACC calves had decayed to nonprotective levels. Calves born to vaccinated dams had a decreased probability of BRSV shedding; however, this was not related to differences in SN or nasal BRSV antibody titers.

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics.

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

Local and Systemic Antibody Responses in Beef Calves Vaccinated with a Modified-Live Virus Bovine Respiratory Syncytial Virus (BRSV) Vaccine at Birth following BRSV Infection.

Maternal antibodies interfere with BRSV vaccine responses and efficacy in young calves. The objective of this study was to determine if vaccination before the complete absorption of colostral antibodies results in adequate immune priming and clinical protection of beef calves. Within 6 h of life, calves were randomly assigned to 2 different treatment groups. Group Vacc (n = 25) received a single dose of a modified-live virus (MLV) BRSV vaccine intranasally (IN) and group Control (n = 25) received 2 mL of 0.9% saline IN. At approximately 3 months of age, all calves were experimentally challenged with BRSV. Serum and nasal secretion samples were collected before and after challenge for BRSV real-time RT-PCR and antibody testing. Respiratory signs were not observed before challenge. After challenge, respiratory scores were similar between groups. On the challenge day, >40% of calves in each group were febrile. The mean serum and nasal BRSV-specific antibody titers indicated natural BRSV exposure before the experimental challenge in both groups. All calves tested positive for BRSV and had a similar duration of shedding after challenge. Based on these results, vaccination at birth does not offer advantages for immune priming or clinical protection for beef calves in BRSV-endemic cow-calf herds.

Evaluation of specific immunoglobulin A in nasal secretions and neutralizing antibodies in serum collected at multiple time points from young beef calves following intranasal or subcutaneous administration of a modified-live bovine respiratory syncytial virus vaccine.

OBJECTIVE: To determine anti-bovine respiratory syncytial virus (BRSV) antibody titers for nasal secretions and serum from beef calves following administration of a modified-live (MLV) BRSV vaccine. ANIMALS: 60 healthy newborn purebred beef calves. PROCEDURES: Calves were randomly assigned to 1 of 3 groups: intranasal (IN)-SC (IN MLV BRSV vaccine within 24 hours of birth and SC MLV BRSV vaccine at 2 months of age), SC-IN (SC MLV BRSV vaccine within 24 hours of birth and IN MLV BRSV vaccine at 2 months of age), or NO-IN (no vaccine within 24 hours of birth and IN MLV BRSV vaccine at 2 months of age). Nasal secretion and serum samples were collected for determination of anti-BRSV antibodies within 24 hours of birth and 2 and 6 months of age. RESULTS: Titers of anti-BRSV IgA antibodies in nasal secretions and BRSV neutralizing antibodies in serum were similar among groups at each sampling time. Within 24 hours of birth, nasal anti-BRSV IgA titers were negligible. At 2 months, mean nasal anti-BRSV IgA titers for calves in IN-SC, SC-IN, and NO-IN groups were 192.84, 224.49, and 114.71, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of anti-BRSV IgA antibodies in the nasal secretions and BRSV neutralizing antibodies in the serum of young beef calves following an MLV BRSV vaccine protocol that consisted of IN or SC vaccine within 24 hours of birth and vice versa at 2 months of age were not different from that following only an IN MLV BRSV vaccine at 2 months of age. However, the lack of any differences may have been attributed to other factors.

Comparison of the immune response following subcutaneous versus intranasal modified-live virus booster vaccination against bovine respiratory disease in pre-weaning beef calves that had received primary vaccination by the intranasal route.

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA).

Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA-LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Lymphatic remodelling in response to lymphatic injury in the hind limbs of sheep.

Contractile activity in the lymphatic vasculature is essential for maintaining fluid balance within organs and tissues. However, the mechanisms by which collecting lymphatics adapt to changes in fluid load and how these adaptations influence lymphatic contractile activity are unknown. Here we report a model of lymphatic injury based on the ligation of one of two parallel lymphatic vessels in the hind limb of sheep and the evaluation of structural and functional changes in the intact, remodelling lymphatic vessel over a 42-day period. We show that the remodelled lymphatic vessel displayed increasing intrinsic contractile frequency, force generation and vessel compliance, as well as decreasing flow-mediated contractile inhibition via the enzyme endothelial nitric oxide synthase. A computational model of a chain of lymphatic contractile segments incorporating these adaptations predicted increases in the flow-generation capacity of the remodelled vessel at the expense of normal mitochondrial function and elevated oxidative stress within the lymphatic muscle. Our findings may inform interventions for mitigating lymphatic muscle fatigue in patients with dysfunctional lymphatics.

Pharmacokinetics of tulathromycin following administration to stocker cattle with remote delivery devices.

Remote delivery devices (RDD) are used by some to administer antimicrobials (AM) to cattle when treatment by manual injection is logistically difficult. However, it is not clear that the pharmacokinetics (PK) of AM administered by RDD is comparable to that for AM administered by injection; thus, it is not certain that cattle treated by RDD experience equivalent AM effect. Fifteen crossbred beef steers (body weight [BW] = 302.5 ± 21.7 kg) were used in a three-way crossover study to determine the PK of tulathromycin following administration with RDD in the BQA injection triangle. Cattle were treated by each of three methods at 2.5 mg of tulathromycin per kg of BW with a 60 d washout period between treatments: 1) subcutaneous injection of tulathromycin (SC), 2) treatment by RDD delivered by air pump projector (AIR, Pneudart, Model 178B) at 4.5 m distance, and 3) treatment by RDD delivered by CO2-powered projector at 7.5 m (CO2, Pneudart, Model 176B). Blood was collected prior to injection and at various points up to 552 h post-administration, pharmacokinetic data were analyzed as a mixed model using animal as a random effect and method of administration, order of administration, and their interaction as fixed effects. Plasma creatine kinase (CK) was measured before treatment and at 24 h after treatment to determine the degree of muscle injury resulting from each treatment. Three darts administered by AIR did not discharge (20%; 95% CI = 4% to 48%); and results from these steers were excluded from analysis. Maximum plasma concentration (718, 702.6, and 755.5 µg/mL for SC, AIR, and CO2, respectively) and area under the concentration-time curve (17,885, 17,423, and 18,796 µg • h/mL for SC, AIR and CO, respectively) were similar and not significantly different between methods of administration. There was an effect of time (P = 0.0002), period (P = 0.0001), and interaction between method of administration and study period (P = 0.0210) on plasma concentration of CK. However, method of treatment (P = 0.6091), interaction between method and time (P = 0.6972), interaction between period and time (P = 0.6153), and 3-way interaction between method, period and time (P = 0.6804) were not different. Results suggest that PK of tulathromycin following delivery by RDD can be similar to subcutaneous injection; however, failure of RDD to discharge after delivery by some types of projectors can cause an important proportion of cattle to fail to receive drug as expected.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. METHODS: Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. RESULTS: Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. CONCLUSION: ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

Influence of sedation on onset and quality of euthanasia in sheep.

The purpose of this study was to determine if dexmedetomidine administered IV prior to euthanasia in sheep affected the speed or quality of euthanasia. Twenty clinically healthy Dorset-cross adult ewes between 1 and 3years of age were enrolled in a randomized blinded experimental trial. The subjects were randomly assigned to receive dexmedetomidine 5µg/kg IV or an equivalent volume of saline. Five minutes later, euthanasia was accomplished with a pentobarbital/phenytoin overdose given IV. The time to apnea, asystole, cessation of audible heartbeat, and absence of corneal reflex were recorded by two blinded investigators. If any muscle spasms, contractions, vocalization, and/or dysrhythmias were noted, the time was recorded and type of ECG abnormality was described. An overall score of the euthanasia event was assigned using a numeric rating scale (NRS) after the animal was declared dead. The time to loss of corneal reflex was significantly longer in sheep given dexmedetomidine compared with those who received saline (P=0.03). Although vocalization was observed only in some animals premedicated with dexmedetomidine, no significance was found for this event and no other significant differences between groups were noted. Dexmedetomidine at 5µg/kg IV 5min prior to injection of pentobarbital/phenytoin for euthanasia did not substantially affect the progress of euthanasia. Dexmedetomidine may be given to sedate sheep prior to euthanasia without concern for it adversely affecting the progress of euthanasia, however vocalization may occur.

Extracorporeal Shockwave Therapy Increases Growth Factor Release from Equine Platelet-Rich Plasma In Vitro.

INTRODUCTION: Extracorporeal shockwave therapy (ESWT) and platelet-rich plasma (PRP) are common treatments for soft tissue injuries in horses. Shockwave triggers cell specific responses to promote healing. Growth factors released from PRP also promote healing. It has been hypothesized that greater growth factor release would amplify the healing process. The combination of ESWT and PRP could promote healing in injured tendons and ligaments in the horse. The objective of this study was to determine if application of shockwaves to PRP samples increases the concentration of transforming growth factor-ß1 (TGF-ß1) and platelet-derived growth factor ßß (PDGF-ßß) released from the platelets in vitro. MATERIALS AND METHODS: PRP was produced from blood drawn from six horses. The PRP from each horse was exposed to the following treatments: (1) positive control (freeze-thaw cycle), (2) untreated negative control, or shockwaves with either (3) a "standard probe" (ESWT-S) with a 2 cm focal width and medium energy density or (4) a "power probe" (ESWT-P) with a 1 cm focal width and high energy density. After each treatment, the samples were centrifuged, and the supernatant was harvested. The supernatant was then used for growth factor quantification via commercially available ELISA kits for TGF-ß1 and PDGF-ßß. RESULTS: Concentrations of TGF-ß1 and PDGF-ßß in PRP that underwent a freeze-thaw cycle were significantly increased compared with all other treatments. Both ESWT-S and ESWT-P resulted in significantly increased TGF-ß1 concentrations, 46 and 33%, respectively, when compared with the negative control. Both ESWT-S and ESWT-P resulted in significantly increased PDGF-ßß concentrations, 219 and 190%, respectively, when compared with the negative control. DISCUSSION: These data indicate that the application of ESWT to PRP increases the expression of growth factors in vitro. This suggests that the combination therapy of local PRP injection followed by ESWT may stimulate release of growth factors from platelets after they have been injected into the area of injury. CONCLUSION: The combination of PRP and ESWT might result in synergism of two modalities previously utilized individually for tendon and ligament injuries in horses.

Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

BACKGROUND: Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. STUDY DESIGN AND METHODS: Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. RESULTS: The collected platelet concentration contained a mean platelet yield of 390 × 103 /µL. Donor platelet count decreased from a mean of 193 × 103 /µL to 138 × 103 /µL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-ß1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. CONCLUSION: Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture.

MRI-Based Assessment of Intralesional Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Equine Tendonitis.

Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 106 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migration, or trilineage differentiation due to the presence of SPIOs. Limbs were imaged on a 1.5-tesla clinical MRI scanner postmortem before and after injection to determine the extent of tendonitis and detect SPIO MSCs. Clusters of labeled cells were visible as signal voids in 6/6 subjects. Coalescing regions of signal void were diffusely present in the peritendinous tissues. Although previous reports have determined that local injury retains cells within a small radius of the site of injection, our study shows greater than expected delocalization and relatively few cells retained within collagenous tendon compared to surrounding fascia. Further work is needed if this is a reality in vivo and to determine if directed intralesional delivery of MSCs is as critical as presently thought.

Determination of the minimum alveolar concentration of isoflurane that blunts adrenergic responses in sheep and evaluation of the effects of fentanyl.

OBJECTIVE: To determine the minimum alveolar concentration that blunts adrenergic responses (MACBAR) for isoflurane and evaluate effects of fentanyl on isoflurane MACBAR in sheep. ANIMALS 13 healthy adult Dorset-cross adult ewes. PROCEDURES: In a crossover design, each ewe was anesthetized 2 times for determination of isoflurane MACBAR. Anesthesia was induced with propofol administered IV. Sheep initially received fentanyl (5 µg/kg, IV, followed by a constant rate infusion of 5 µg/kg/h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment). After a washout period of at least 8 days, the other treatment was administered. For MACBAR determination, a mechanical nociceptive stimulus (ie, sponge forceps) was applied at the coronary band for 1 minute. The MACBAR values of the 2 treatments were compared by means of a paired t test. During MACBAR determination, blood samples were collected for measurement of plasma fentanyl concentration. RESULTS: Mean ± SD isoflurane MACBAR of the fentanyl and control treatments was 1.70 ± 0.28% and 1.79 ± 0.35%, respectively; no significant difference was found between the 2 treatments. Plasma concentration of fentanyl reached a median steady-state concentration of 1.69 ng/mL (interquartile range [25th to 75th percentile], 1.47 to 1.79 ng/mL), which was maintained throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of fentanyl at 5 µg/kg, IV, followed by a constant rate infusion of the drug at 5 µg/kg/h did not decrease isoflurane MACBAR. Further studies to determine the effect of higher doses of fentanyl on inhalation anesthetic agents and their potential adverse effects are warranted.

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.