Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices.

Introduction: Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy. Methods: Plasma samples were obtained from healthy adult horses (N = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected reaction monitoring measured a specific A2M peptide as a surrogate of the whole A2M protein. A2M concentrations produced by each test device were compared for two sample types: a pre-concentrate or platelet-poor (PP) component and a final component for use in the horse. Results: There was no significant difference (p > 0.05) in the geometric mean (GM) concentration of A2M in the final concentration samples produced by the Alpha2EQ® device (N horses = 13) and the single-centrifugation PP samples produced by the Pro-Stride® APS (autologous protein solution) device (N = 13) and the Restigen® PRP (platelet-rich plasma) device (N = 11). When A2M content in final concentration samples produced by each device was compared, the Pro-Stride APS and Restigen PRP samples had significantly greater GM A2M content (p < 0.0001) compared to the Alpha2EQ samples, and the Pro-Stride APS final concentration samples had significantly greater GM A2M concentration (p < 0.0001) versus that for the Restigen PRP final samples. Discussion: This comparison demonstrated that the volume and A2M concentration of an Alpha2EQ final concentrate are no different than the volume and concentration of A2M in the PP from Pro-Stride or Restigen devices.

Utility of serum amyloid A in monitoring clinical response to antimicrobial treatment in horses with bacterial pneumonia.

BACKGROUND: Serum amyloid A (SAA) is a major acute phase protein in horses which could be a useful tool for assessing clinical response to treatment of bacterial pneumonia in adult horses. OBJECTIVES: To monitor SAA concentration in response to treatment and identify associations among SAA concentration, WBC and neutrophil counts, and fibrinogen in bacterial pneumonia in adult horses. ANIMALS: Eighteen adult horses with bacterial pneumonia. METHODS: Prospective clinical study. Horses hospitalized with bacterial pneumonia were enrolled and SAA concentration and vital signs were assessed daily. SAA concentration was measured by a handheld meter. CBC and plasma fibrinogen were assessed on days 0, 1, and 2, then every 3 days until discharge. Data were not normally distributed and therefore were log transformed. Log-transformed data were analyzed and comparisons were performed on LSMeans by the 2-sided Student's t-test at the 5% level of significance. RESULTS: Geometric mean SAA concentration on day 0 was 537 µg/mL (SE 383 µg/mL). Geometric mean SAA concentration decreased significantly over time (P = .0001), peaking at day 2 (geomean 1038 µg/mL, SE 261.7 µg/mL) and decreasing until discharge. Plasma concentration of fibrinogen (P = .06), neutrophil count (P = .48), and WBC count (P = .07) did not change significantly over time. CONCLUSIONS AND CLINICAL IMPORTANCE: SAA concentration decreased significantly over the course of treatment and correlated with clinical improvement of pneumonia whereas fibrinogen, neutrophil, and WBC counts did not.

Investigation of the Use of Serum Amyloid A to Monitor the Health of Recently Imported Horses to the USA.

The objective of this study was to investigate the use of serum amyloid A (SAA) as an early indicator of subclinical inflammation in recently imported horses. Archived serum samples from 143 adult horses imported from Europe over 12 months were available for SAA testing. Based on clinical and hematological assessment performed shortly after arrival to a quarantine facility, the horses were characterized as healthy horses, horses with subclinical inflammation, and sick horses with and without hematological abnormalities. The majority of the horses (n = 109) were deemed healthy, 30 horses had evidence of subclinical inflammation based on hematological abnormalities, and 4 horses were sick. SAA values ranged from 0 to 3,000 µg/mL (median 9 µg/mL) in healthy horses and from 0 to 1,522 µg/mL (median 9 µg/mL) in horses with subclinical inflammation, while 3 out of 4 sick horses had elevated SAA values (range 15-1,923 µg/mL, median 590 µg/mL). The cause for the elevated SAA values in the majority of the healthy horses and horses with subclinical inflammation could not be determined. Overall, a single point in time SAA test did not add additional value to routine clinical and hematological monitoring in recently imported horses.

Investigation of The Usefulness of Serum Amyloid A in Characterizing Selected Disease Forms of Equine Herpesvirus-1 Infection.

The objective of this study was to study the SAA response of horses with various forms of EHV-1 infection. Archived serum samples from 153 horses with various disease forms of EHV-1 infection (48 healthy non-infected horses, 48 subclinically infected horses, 40 horses with respiratory EHV-1 infection and 17 horses with neurological EHV-1 infection) were available for SAA testing. SAA values ranged from 0 to 31 µg/mL (median 0 µg/mL) in healthy horses, from 0 to 2,416 µg/mL (median 8.5 µg/mL) in subclinically infected horses, from 0 to 3,000 µg/mL (median 597 µg/mL) in horse with respiratory EHV-1 infection and from 0 to 1,640 µg/mL (median 58 µg/mL) in horse with neurological EHV-1 disease. Infected horses had significantly higher SAA values compared to healthy, non-infected horses. While SAA was elevated in the majority of horses with evidence of EHV-1 infection, a single point in time SAA test was unable to consistently support infection in horses with subclinical disease.

Reproductive, productivity, and mortality outcomes in late-gestation gilts and their litters following simulation of inadvertent exposure to a modified-live vaccine strain of porcine reproductive and respiratory syndrome (PRRS) virus.

The study evaluated the safety of a modified live-virus (MLV) porcine reproductive and respiratory syndrome (PRRS) vaccine in susceptible, pregnant gilts. To simulate inadvertent exposure secondary to postvaccination shedding of PRRS-MLV, seronegative gilts (n=51) were exposed by IM vaccination at 90 days of gestation. Vaccinated and nonvaccinated, seronegative control gilts (n=25) were maintained in separate facilities. The PRRS-MLV vaccine was given in a 2mL dose on day 0. On day 7 all vaccinated gilts were PRRSV-PCR-positive for PRRSV and had responded serologically as determined by an ELISA. All control gilts remained PRRSV-PCR- and ELISA-negative throughout the study. Abortions did not occur in gilts from either group. The difference between vaccinated and control gilts in average number of piglets per litter (12.43 and 12.16, respectively), number of live births per litter (11.21 and 11.54), and mean piglet birth weight (3.22 and 3.26 lbs) were not significantly different. Piglets in the control group had significantly greater average daily gain versus piglets from vaccinated gilts (0.52 vs. 0.46 lbs, P<0.0001). Preweaning mortality was significantly greater (P=0.0023) in piglets from the vaccinated gilts (19.7% vs. 10.9%). A single gilt accounted for 18.2% of stillbirths in the vaccinated group. Air samples were borderline PRRSV-PCR-positive for PRRSV on days 29 and 32, after more than 98% of gilts had farrowed. Results demonstrated that vaccination of pregnant gilts at the time of peak fetal susceptibility was non-abortigenic and that the PRRS-MLV agent did not significantly affect reproductive outcomes. Lower ADG in piglets from vaccinated gilts may be due to PRRS-MLV viremia following transplacental or post-farrowing exposure. Air sampling results indicated that environmental contamination with PRRS-MLV shed from vaccinated gilts was minimal.