Evaluation of post-transfusion RBC alloimmunization in dogs using a gel-column crossmatch with and without anti-canine globulin enhancement.

A blood crossmatch is essential to ensure RBC compatibility for previously transfused dogs. There is no gold standard crossmatch method for dogs, although the standards used most commonly by academic institutions and reference laboratories are the tube and gel-column crossmatches. Addition of anti-canine globulin (ACG) has been suggested to increase detection of RBC incompatibilities. Our objective was to determine if there is a correlation between results of a standard and an ACG-enhanced gel-column crossmatch in detecting post-transfusion RBC alloimmunization. Pre- and post-transfusion serum or plasma samples were obtained from 33 dogs for major crossmatches to 1-6 (median: 3) blood donors. Crossmatches were performed with (n = 202) and without (n = 202) ACG, with results scored by 4 observers, 3 of whom were anonymized. Ten of 33 (30%) dogs had major crossmatch incompatibilities post-transfusion. RBC incompatibilities (2-4+ agglutination) were detected only with ACG in 4 dogs, only without ACG in 3 dogs, and with both methods in 3 dogs. There was fair correlation between crossmatch methods for determination of compatibility (ρ = 0.34; p < 0.001) and incompatibility (ρ = 0.35; p < 0.001) scores. Among 4 observers, there was near-perfect agreement in determining compatibility (κ = 0.97; p < 0.001) and substantial agreement in overall scoring of incompatibility (κ = 0.77; p < 0.001). Our results suggest that detection of RBC incompatibilities in dogs can be maximized by performing a gel-column crossmatch both with and without ACG enhancement.

An Evaluation of Messages to Promote Parental Intent to Vaccinate Children Aged <12 Years Against COVID-19.

OBJECTIVES: Effective health communication can increase intent to vaccinate. We compared 8 messages that may influence parents' intent to vaccinate their children against COVID-19. METHODS: In a cross-sectional survey of adults in the United States administered online in August 2021, 1837 parents and legal guardians were exposed to 8 messages (individual choice, gain/practical benefits, nonexpert, health care provider recommendation, altruism/community good, safety/effectiveness, safety, and effectiveness) to determine message reception and influence on intent to vaccinate their children. Parents responded to 10 questions using a Likert scale. We computed odds ratios (ORs) for each message, with an OR >1.0 indicating greater observed odds of participant agreement with the follow-up statement as compared with a reference message. We compared outcomes individually across messages with ordinal logistic regression fit using generalized estimating equations. RESULTS: The individual choice message had the highest odds of agreement for understanding intent (OR = 2.10; 95% CI, 1.94-2.27), followed by the health care provider recommendation message (OR = 1.58; 95% CI, 1.46-1.71). The individual choice message had the highest odds of memorability, relatability, and trustworthiness. The altruism/community good message was at or near second best. The altruism/community good message had the highest or near-highest odds of increasing parents' intent to vaccinate their children, asking friends and family for their thoughts, and searching for additional information. The message that most motivated parents to vaccinate their children depended on parental intent to vaccinate prior to being exposed to the tested messages. CONCLUSIONS: Messages with themes of individual choice, health care provider recommendation, and altruism/community good may be used in future message campaigns. Further research is needed to refine message concepts related to altruism/community good.

COVID-19 Vaccine Hesitancy Among US Adults: Safety and Effectiveness Perceptions and Messaging to Increase Vaccine Confidence and Intent to Vaccinate.

OBJECTIVE: Public health agencies have a critical role in providing effective messaging about mitigation strategies during a public health emergency. The objectives of this study were (1) to understand perceptions of COVID-19 vaccines, including concerns about side effects, safety, and effectiveness and how these perceptions influence vaccine decision-making among US adults and (2) to learn what messages might motivate vaccine uptake. METHODS: In April and May 2021, we conducted 14 online focus groups with non-Hispanic English-speaking and English- and Spanish-speaking Hispanic adults (N = 99) not vaccinated against COVID-19. We oversampled adults aged 18-39 years and rural residents and systematically assessed 10 test messages. Researchers used a standardized guide and an a priori codebook for focus group discussions, coding transcripts, and thematic analysis. RESULTS: Vaccine hesitancy factors included fear of the unknown; long-term side effects, including infertility; and beliefs that the vaccines were developed too quickly and were not sufficiently effective. Motivating factors for receiving vaccination included the ability to safely socialize and travel. Health care providers were considered important trusted messengers. Participants were critical of most messages tested. Messages that came across as "honest" about what is not yet known about COVID-19 vaccines were perceived more positively than other messages tested. Messages were seen as ineffective if perceived as vague or lacking in data and specificity. CONCLUSIONS: Messages that were simple and transparent about what is unknown about vaccines relative to emerging science were viewed most favorably. Health care providers, friends, and family were considered influential in vaccination decision-making. Findings underscore the benefits of research-informed strategies for developing and disseminating effective messages addressing critical issues in a public health emergency.

Effect of donor blood storage on gel column crossmatch in dogs.

BACKGROUND: Compared with fresh blood, stored equine donor blood results in spurious tube crossmatch incompatibilities. Interpretation of blood crossmatch results is considered subjective. OBJECTIVES: We aimed to determine if the duration of canine donor blood storage impacts compatibility testing using a standard gel column crossmatch and evaluate interobserver variation in the interpretation of crossmatch results. METHODS: Observational study. Whole blood segments were obtained from 23 canine packed red blood cell (RBC) units for use in crossmatches after storage for 0, 7, 14, 21, 28, and 35 days. Major and minor crossmatches were performed using serum and RBCs, respectively, from two to three healthy "recipient" dogs per unit. All crossmatch results were interpreted by four observers, of whom three were blinded. RESULTS: All major crossmatches (n = 61) were compatible on day 0 and remained compatible through day 35 of storage. All minor crossmatches (n = 69) were compatible at all time points, except for five donor pairs with 1 to 3+ agglutination. Repeat testing of these five donor pairs confirmed crossmatch incompatibilities on days 0 through 35, with no change in the degree of incompatibility over time. There was substantial agreement among four observers in determining compatibility (κ = 0.94) and scoring incompatibility (κ = 0.76). CONCLUSIONS: The current practice of performing canine crossmatches with whole blood segments stored for up to 35 days is acceptable, with no spurious changes in compatibility expected over time. The substantial interobserver agreement suggests that the gel column is suitable for performing canine crossmatches in a laboratory setting with multiple personnel.

Clinical application of the StatSensor and StatSensor Xpress point-of-care creatinine measurement devices in dogs.

BACKGROUND: Creatinine is a universally important blood parameter used to detect and monitor acute and chronic kidney disease. Reliable measurements at the bedside remain a challenge in human and veterinary medicine. Despite its potential, a trustworthy point-of-care creatinine biosensor has yet to be established. OBJECTIVES: We aimed to determine the precision and accuracy of the StatSensor (SS) and StatSensor Xpress (SSX) handheld creatinine measurement devices in dogs. METHODS: Paired creatinine samples from dogs with normal (creatinine ≤159 µmol/L), moderate (159-354 µmol/L), and marked (>354 µmol/L) azotemia were compared with a commercial enzymatic analyzer. Within-day precision and linearity studies were performed prior to method comparison studies. Method comparison was evaluated using Bland-Altman, concordance correlation coefficient, Deming, and Passing-Bablok regression analysis. RESULTS: Seventy-eight dogs were enrolled in the study, including 28 (35%), 25 (32%), and 26 (33%) with normal, moderate, and marked azotemia. Total error surpassed recommendations for all devices, and linearity deviated from identity for the SS1 and SS2. The concordance correlation coefficients of the SS1, SS2, SSXI, and SSX2, were 0.69, 0.59, 0.82, and 0.44, respectively. Bland-Altman analyses showed a high variation in the differences, and relationships showed high heteroskedasticity with negative systemic bias among high creatinine concentrations. CONCLUSIONS: Neither the SS and SSX are considered acceptable for clinical applications in dogs. Further research is indicated for the development of a reliable, cost-effective, point-of-care creatinine analyzer to improve the rapid detection and monitoring human and veterinary patients.

Response-based modification of CHOP chemotherapy for canine B-cell lymphoma.

Despite high initial response rates, a subset of dogs with B-cell lymphoma responds less robustly to CHOP-based chemotherapy and experiences shorter survival. One hundred and four dogs with nodal B-cell lymphoma were treated with a response-based CHOP (RBCHOP) protocol modified based on response to individual drugs during the first chemotherapy cycle. Dogs achieving complete (CR) or partial response (PR) at week 3, following treatment with vincristine and cyclophosphamide, received RBCHOP 1 (n = 72), a protocol sequentially rotating vincristine, cyclophosphamide, and doxorubicin. Dogs without a detectable response at week 3 that subsequently achieved CR or PR following treatment with doxorubicin received RBCHOP 2 (n = 14), in which four doses of doxorubicin were given consecutively followed by vincristine and cyclophosphamide. Dogs that failed to respond at week 3 and then to doxorubicin at week 5 assessment were offered rescue chemotherapy (RBCHOP 3, n = 18). Median progression free survival (PFS) and overall survival time (OST) were similar between RBCHOP 1 (PFS 210 days, OST 354 days) and RBCHOP 2 (PFS 220 days, OST 456 days), but significantly shorter for RBCHOP 3 (PFS 34 days, OST 80.5 days, P < 0.001). No presenting signalment nor hematologic variable differentiated patient cohort, however, dogs in RBCHOP 2 and RBCHOP 3 were more likely to have a lymphocytosis at diagnosis (P = 0.02 and 0.04, respectively). Protocol modification based on response during the first cycle resulted in similar toxicity profiles and outcomes to previously published variants of CHOP, and prognosis remained poor for dogs failing to respond during the first treatment cycle.

Urine cortisol-creatinine and protein-creatinine ratios in urine samples from healthy dogs collected at home and in hospital.

BACKGROUND: Recently, urine protein:creatinine ratios (UPC) were shown to be lower in urine samples from dogs collected at home (AH) as compared to those collected in hospital (IH). Stress-inducing procedures and travel to the hospital have been hypothesized to cause prerenal proteinuria. OBJECTIVES: Evaluate patient stress using urine cortisol:creatinine ratios (UCCr) and correlate UCCr to UPC in urine samples obtained AH and IH. ANIMALS: Thirty-six healthy, client-owned dogs. METHODS: Prospective, non-masked study. Two voided urine samples were obtained (AH and IH). Complete urinalysis as well as UPC and UCCr were performed. Clients graded their dogs' stress level AH, in transport, and IH. RESULTS: The UCCr was significantly higher in IH samples than in AH samples (P < .0001), but UPC was not significantly different between AH and IH urine samples (P = .14). In all samples and in both collection settings, UCCr was not significantly correlated with UPC. Travel time and time IH were not correlated with change in UCCr or UPC. In 8 dogs with borderline or overt proteinuria, no significant difference was found in UPC between settings, but UCCr was significantly higher in IH samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The UPC was not higher when measured in urine samples collected IH compared to AH. Dogs had higher UCCr IH, but UCCr was not associated with UPC. Stress, as estimated by UCCr, did not affect proteinuria. Further evidence is needed to support the claim that stress may result in proteinuria in healthy dogs.

Toxicity and Efficacy Evaluation of an Adeno-Associated Virus Vector Expressing Codon-Optimized RPGR Delivered by Subretinal Injection in a Canine Model of X-linked Retinitis Pigmentosa.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated rAAV2tYF-GRK1-hRPGRco, to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (hRPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1 [GRK1]), and is packaged in an AAV2 capsid variant with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a toxicity and efficacy study of this vector administered by subretinal injection in the naturally occurring RPGR mutant (X-linked progressive retinal atrophy 2 [XLPRA2]) dog model. Sixteen RPGR mutant dogs divided into four groups of three to five animals each received either a subretinal injection of 0.07 mL of AGTC-501 at low (1.2 × 1011 vector genome [vg]/mL), mid (6 × 1011 vg/mL), or high dose (3 × 1012 vg/mL), or of vehicle control in the right eye at early-stage disease. The left eye remained untreated. Subretinal injections were well tolerated and were not associated with systemic toxicity. Electroretinography, in vivo retinal imaging, and histological analysis showed rescue of photoreceptor function and structure in the absence of ocular toxicity in the low- and mid-dose treatment groups when compared with the vehicle-treated group. The high-dose group showed evidence of both photoreceptor rescue and posterior segment toxicity. These results support the use of AGTC-501 in clinical studies with patients affected with XLRP caused by RPGR mutations and define the no-observed-adverse-effect level at 6 × 1011 vg/mL.

Prenatal Practice Staff Perceptions of Three Substance Use Screening Tools for Pregnant Women.

OBJECTIVE: There is a need to identify an acceptable and comprehensive substance use screening tool for pregnant women in the United States. This qualitative study sought to better understand prenatal practice staff perceptions of three existing substance use screening tools for use among pregnant women in an outpatient practice setting. METHODS: Eight focus groups with 40 total participants were conducted with clinical and administrative staff of 2 diverse Maryland prenatal practices to determine the acceptability and usability of 3 substance use screening tools (4P's Plus, NIDA-Modified Alcohol, Smoking and Substance Involvement Screening Test, and the Substance Use Risk Profile-Pregnancy scale). The focus groups were digitally recorded, transcribed, coded, and analyzed using thematic analysis. RESULTS: Participant perceptions of screening tools were dependent upon screening tool length, tone, comprehensiveness, subjectivity, time frame of questions, and scoring and clinician instructions. Most participants preferred the 4P's Plus screening tool because it is brief, comprehensive, easy for the patient to understand, and excludes judgmental language and subjective questions. CONCLUSIONS: These results provide valuable insight into the specific needs and preferences of prenatal practice staff as it relates to prenatal substance use screening and provides evidence that the 4P's Plus may be a preferred screening tool for standardized use in prenatal care.

Prevalence of naturally occurring non-AB blood type incompatibilities in cats and influence of crossmatch on transfusion outcomes.

BACKGROUND: Recognition of the feline red blood cell (RBC) antigen Mik and the presence of naturally occurring anti-Mik antibodies resulting in acute hemolytic transfusion reactions prompted the recommendation to perform a crossmatch before a cat's first RBC transfusion, but this guideline has not yet become a standard practice. OBJECTIVE: To determine the prevalence of naturally occurring non-AB alloantibodies detectable by tube crossmatch, and to compare transfusion outcomes in cats with and without a crossmatch performed. ANIMALS: Three hundred cats that received an RBC transfusion, with or without a major crossmatch performed. METHODS: Retrospective study. RESULTS: Major crossmatch incompatibilities were documented in 23 of 154 transfusion-naive cats (14.9%) and in 15 of 55 previously transfused cats (27%; P = 0.042). Type-specific packed RBCs (pRBCs) were administered to 167 and 82 cats with and without a crossmatch, respectively. Median volume of pRBCs administered during the first transfusion was 5.3 mL/kg (range, 2.4-18 mL/kg). Median change in PCV scaled to dose of pRBCs was +0.8%/mL/kg; administration of crossmatch-compatible pRBCs was not associated with a greater increase in PCV. Febrile transfusion reactions occurred more often in cats that received non-crossmatched (10.1%) compared to crossmatched (2.5%) pRBCs (P = 0.022). Seventy-six percent of cats that received pRBC transfusions survived to hospital discharge. A crossmatch was not associated with improved survival to discharge or at 30 or 60 days posttransfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: The prevalence of naturally occurring non-AB incompatibilities is sufficiently high to justify the recommendation to perform a crossmatch before all (including the first) RBC transfusions in cats.

Higher plasma corticosterone is associated with reduced costs of infection in red-winged blackbirds.

Glucocorticoid hormones allow individuals to rapidly adjust their physiology and behavior to meet the challenges of a variable environment. An individual's baseline concentration of glucocorticoids can reflect shifts in life history stage and resource demands while mediating a suite of physiological and behavioral changes that include immune modulation and resource allocation. Thus, glucocorticoids could facilitate a response to parasites that is optimized for an individual's specific challenges and life history stage. We investigated the relationship between endogenous circulating glucocorticoids and measures of resistance and tolerance to Haemosporidian parasites (including those that cause avian malaria) in red-winged blackbirds (Agelaius phoeniceus). We found that higher endogenous concentrations of circulating glucocorticoids were associated with reduced costs of parasite infection, which is indicative of higher tolerance, but were unrelated to parasite burden in free ranging, breeding male birds. Post-breeding, both males and females with higher glucocorticoid concentrations had higher measures of tolerance to Haemosporidian infection. Our findings suggest a potentially adaptive role for glucocorticoids in shifting the response to parasites to align with an individual's current physiological state and the challenges they face.

Testosterone and Haemosporidian Parasites Along a Tropical Elevational Gradient in Rufous-Collared Sparrows (Zonotrichia capensis).

Elevation has been proposed as a dominant ecological variable shaping life history traits and subsequently their underlying hormonal mechanisms. In an earlier meta-analysis of tropical birds, elevation was positively related to testosterone levels. Furthermore, parasitism by avian haemosporidians should vary with elevation as environmental conditions affect vector abundance, and while testosterone is needed for breeding, it is hypothesized to be immunosuppressive and thus could exacerbate haemosporidian infection. Our objective in this study was to examine the relationships between elevation, testosterone levels, and parasitism by avian haemosporidians. We surveyed breeding male rufous-collared sparrows (Zonotrichia capensis) across a wide elevational range along the equator. We measured baseline testosterone levels, haemosporidian infection at four elevations spanning the species' natural range in the Ecuadorian Andes (600, 1500, 2100, 3300 m). Testosterone levels from breeding males were not related to elevation, but there was high intrapopulation variability. Testosterone levels were not related to the probability of parasitism, but our results from one population suggested that the likelihood of being infected by haemosporidian parasites was greater when in breeding condition. In conclusion, even though there is variation in life history strategies among the studied populations, wider divergence in seasonality and life history traits would probably be needed to detect an effect of elevation on testosterone if one exists. Additionally, our results show that variation in testosterone is not related to infection risk of haemosporidians, thus other factors that take a toll on energetic resources, such as reproduction, should be looked at more closely.

Meningeal norepinephrine produces headache behaviors in rats via actions both on dural afferents and fibroblasts.

BACKGROUND: Stress is commonly reported to contribute to migraine although mechanisms by which this may occur are not fully known. The purpose of these studies was to examine whether norepinephrine (NE), the primary sympathetic efferent transmitter, acts on processes in the meninges that may contribute to the pain of migraine. METHODS: NE was applied to rat dura using a behavioral model of headache. Primary cultures of rat trigeminal ganglia retrogradely labeled from the dura mater and of rat dural fibroblasts were prepared. Patch-clamp electrophysiology, Western blot, and ELISA were performed to examine the effects of NE. Conditioned media from NE-treated fibroblast cultures was applied to the dura using the behavioral headache model. RESULTS: Dural injection both of NE and media from NE-stimulated fibroblasts caused cutaneous facial and hindpaw allodynia in awake rats. NE application to cultured dural afferents increased action potential firing in response to current injections. Application of NE to dural fibroblasts increased phosphorylation of ERK and caused the release of interleukin-6 (IL-6). CONCLUSIONS: These data demonstrate that NE can contribute to pro-nociceptive signaling from the meninges via actions on dural afferents and dural fibroblasts. Together, these actions of NE may contribute to the headache phase of migraine.

Dural fibroblasts play a potential role in headache pathophysiology.

Nociceptive signaling from the meninges is proposed to contribute to many forms of headache. However, the events within the meninges that drive afferent activity are not clear. Meningeal fibroblasts are traditionally thought to produce extracellular proteins that constitute the meninges but not to contribute to headache. The purpose of these studies was to determine whether dural fibroblasts release factors that activate/sensitize dural afferents and produce headache-like behavior in rats. Dura mater was removed from male rats and dural fibroblasts were cultured. Fibroblast cultures were stimulated with vehicle or lipopolysaccharide (LPS), washed, and conditioned media was collected. Fibroblast media conditioned with vehicle or LPS was applied to retrogradely labeled rat dural trigeminal ganglion neurons in vitro. Patch-clamp electrophysiology was performed to determine whether conditioned media activated/sensitized dural afferents. A preclinical behavioral model was used where conditioned media was applied directly to the rat dura to determine the presence of cutaneous facial and hind-paw allodynia. Conditioned media was also tested for interleukin-6 (IL-6) content using an enzyme-linked immunosorbent assay. Application of LPS-conditioned fibroblast media to dural afferents produced a significant increase in action potential firing as well as cutaneous facial and hind-paw allodynia when this media was applied to the dura. Finally, stimulation of cultured fibroblasts with LPS increased IL-6 levels in the media. These findings demonstrate that fibroblasts stimulated with LPS release factors capable of activating/sensitizing dural afferents. Further, they suggest that fibroblasts play a potential role in the pathophysiology of headache.

Variation in body condition indices of crimson finches by sex, breeding stage, age, time of day, and year.

Body condition indices are increasingly applied in conservation to assess habitat quality, identify stressed populations before they decline, determine effects of disturbances, and understand mechanisms of declines. To employ condition indices in this manner, we need first to understand their baseline variability and sources of variation. Here, we used crimson finches (Neochmia phaeton), a tropical passerine, to describe the variation in seven commonly used condition indices by sex, age, breeding stage, time of day, and year. We found that packed cell volume, haemoglobin, total plasma protein, and scaled mass were all significantly affected by an interaction between sex and breeding stage. Furcular fat varied by sex and breeding stage and also trended by year, scaled mass showed a positive trend with age and varied by time of day, and haemoglobin additionally varied by year. Pectoral muscle scores varied and heterophil to lymphocyte ratio trended only by year. Year effects might reflect a response to annual variation in environmental conditions; therefore, those indices showing year effects may be especially worthy of further investigation of their potential for conservation applications. Pectoral muscle scores and heterophil to lymphocyte ratio may be particularly useful due to the lack of influence of other variables on them. For the other indices, the large variation that can be attributed to individual covariates, such as sex and breeding stage, suggests that one should not interpret the physiological condition of an individual as measured by these indices from their absolute value. Instead, the condition of an individual should be interpreted relative to conspecifics by sex, breeding stage, and possibly age.

Effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs.

OBJECTIVE: To determine the effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs and to assess intraindividual variation in thromboelastography results. ANIMALS: 14 healthy research dogs and 10 healthy client-owned dogs. PROCEDURES: In a randomized controlled trial, research dogs underwent thromboelastography twice (3 days apart), and intraindividual variation in test results was calculated. Dogs were given prednisone (2 mg/kg/d, PO) plus acetylsalicylic acid (0.5 mg/kg/d, PO) or prednisone (2 mg/kg/d, PO) plus a placebo for 14 days, after which thromboelastography and other tests were repeated. Differences from preadministration (baseline) test results between and within groups were compared. In a separate trial, client-owned dogs also underwent thromboelastography twice 2 days apart to assess intraindividual variation in untreated dogs. RESULTS: Intraindividual variation in thromboelastography results for research dogs was ≤ 10% for maximum amplitude (MA) and α angle. In the research dogs, MA and fibrinogen values significantly increased from baseline, whereas percentage lysis 30 minutes after attainment of the MA as well as antithrombin activity significantly decreased within each group. In the dogs that received prednisone plus a placebo, percentage lysis 60 minutes after attainment of the MA was significantly lower than at baseline. For all parameters for research dogs, there was no difference between groups for change from baseline. Intraindividual variation in findings for client-owned dogs was similar to the variation for research dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Prednisone administration resulted in hypercoagulability in healthy dogs as indicated by an increase in MA and plasma fibrinogen concentration and a decrease in antithrombin activity. Concurrent ultralow-dose acetylsalicylic acid use had no effect on measured thromboelastography values. The high intraindividual variation in some thromboelastography parameters may preclude routine use of this technique in clinical practice.