Thromboelastographic Clot Characteristics of Autologous Equine Blood Products After Activation by Autologous Thrombin,Bovine Thrombin,or Calcium Chloride

Objective

To compare clotting efficiency of platelet‐rich plasma (PRP) and concentrated platelet‐poor plasma (cPPP) to citrated whole blood after activation by autologous thrombin, bovine thrombin, or calcium chloride (CaCl₂).

Study Design

Experimental study.

Animals

Healthy adult horses (n = 6).

Methods

PRP and cPPP were prepared by commercial devices. Using thromboelastography, clotting variables were compared after activation of citrated autologous blood, PRP, and cPPP by autologous thrombin, bovine thrombin, or CaCl₂, respectively.

Results

PRP had the greatest clot strength and quickest clot rate, whereas cPPP had the weakest clot strength, slowest clot rate, and longest clot initiation time. Bovine thrombin resulted in the shortest clot initiation time, quickest clot rate, and was similar to CaCl₂ for greatest clot strength. CaCl₂ also resulted in the longest clot initiation time and time to reach maximum clot strength. Autologous thrombin resulted in the lowest clot strength.

Conclusion

When combined with either bovine thrombin or CaCl₂, PRP provided the best combinations for clinical use. Autologous thrombin was suboptimal, but could be an autologous alternative for clinical application. As prepared here, cPPP had inefficient clotting, but may be sufficient for plasma spray indications.     

Bacterial Contamination of Stethoscope Chest Pieces and the Effect of Daily Cleaning

Background

Stethoscopes are a potential source of nosocomial infection for hospitalized humans, a phenomenon not previously studied in companion animals.

Objectives

To determine if daily cleaning of stethoscope chest pieces reduces bacterial contamination between cleanings.

Animals

Client‐owned dogs and cats.

Methods

Prospective observational study. In phase 1, bacterial cultures were obtained from the chest pieces of 10 participant stethoscopes once weekly for 3 weeks. In phase 2, stethoscopes were cleaned daily and 2 culture samples were obtained once weekly, immediately before and after cleaning with 70% isopropyl alcohol, for 3 weeks.

Results

Daily cleaning eliminated bacteria immediately after each cleaning (P = .004), but did not reduce the rate of positive cultures obtained before cleaning in phase 2. Cultures were positive for 20/30 (67%) samples during phase 1 and 18/30 (60%) obtained before daily cleaning during phase 2. Recovered organisms included normal skin flora, agents of opportunistic infections, and potential pathogens. The only genus that was repeatedly recovered from the same stethoscope for 2 or more consecutive weeks was Bacillus sp.

Conclusions and Clinical Importance

Daily cleaning was highly effective at removing bacteria, but provided no reduction in precleaning contamination. Cleaning stethoscopes after use on dogs or cats infected with pathogenic bacteria and before use on immunocompromised animals should be considered.     

Comparison of Multiplate,Platelet Function Analyzer‐200, and Plateletworks in Healthy Dogs Treated with Aspirin and Clopidogrel

Background

Platelet function testing may be warranted to assess response to aspirin and clopidogrel.

Hypothesis/Objectives

To evaluate the effects of aspirin, clopidogrel, or combination therapy using 3 platelet function tests: Multiplate Analyzer (MP), Platelet Function Analyzer‐200 (PFA), and Plateletworks (PW).

Animals

Six healthy laboratory Beagles.

Methods

Randomized double‐blind placebo‐controlled study (crossover design). Dogs were given aspirin 1 mg/kg, clopidogrel 2 mg/kg, or combination therapy for 1 week each, with a washout period of 2 weeks. Platelet function was assessed on days 0 and 7 of each phase using MP (adenosine diphosphate [ADP], arachidonic acid [AA], collagen [COL] agonists), PFA (P2Y, COL‐ADP [CADP], COL‐Epinephrine [CEPI] cartridges), and PW (ADP, AA, COL agonists). Platelet counts were obtained with impedance and optical counters.

Results

For MP, mean aggregation was decreased for COL and AA with combination therapy and for ADP with all treatments. For PFA, mean CT was increased for the CEPI cartridge with aspirin; and for the P2Y and CADP cartridges with clopidogrel or combination therapy. More dogs receiving clopidogrel showed an increase in PFA CT using the P2Y than the CADP cartridge. For PW, mean aggregation was decreased for AA with all treatments; for ADP with clopidogrel or combination therapy; and for COL with clopidogrel. The PW results with the 2 hematology counters showed almost perfect agreement.

Conclusion and Clinical Importance

All platelet function tests detected treatment effects in some dogs and may have utility for monitoring therapy.     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Normal reference intervals and the effects of sample handling on dynamic viscoelastic coagulometry (Sonoclot) in healthy adult horses

Objective

To determine reference intervals and the effect of sample agitation and rest time on Sonoclot analysis in healthy adult horses.

Design

Original prospective study.

Setting

University veterinary medical teaching hospital.

Animals

Sixty healthy adult horses.

Interventions

Blood was collected for assessment of complete blood count, serum biochemical analysis, and Sonoclot analysis.

Measurements and Main Results

Horses were determined to be healthy based upon physical examination, CBC, and serum biochemistry analysis. Blood was analyzed in a glass bead‐containing cuvette using the Sienco Sonoclot analyzer following 2 rest periods (30 mins and 240 min) and with 2 sample handling interventions (agitated and nonagitated), to obtain values for clot rate, time‐to‐peak, activated clotting time, and platelet function. This study failed to detect a significant difference when a rest time of 30 minutes was compared with 240 minutes, but based on wide limits of agreement the 2 rest times were not considered interchangeable. Agitation at both rest times significantly affected all Sonoclot analyses leading to changes indicative of hypercoagulability.

Conclusions

Sample agitation and rest time should be taken into consideration when developing preanalytical guidelines for Sonoclot analysis in horses. Calculated reference intervals were relatively wide. Further research is needed to evaluate the clinical utility of Sonoclot analysis in horses.     

Echocardiographic Estimates of Right Ventricular Systolic Function in Dogs with Myxomatous Mitral Valve Disease

Background

Right ventricular (RV) dysfunction independently predicts outcomes in human myxomatous mitral valve disease (MMVD). There is limited information regarding RV systolic function in dogs with MMVD.

Hypothesis

Right ventricular systolic function differs among stages of disease, decreasing in decompensated MMVD.

Animals

Thirty‐sixclient‐owned dogs with MMVD not receiving oral cardiovascular medications.

Methods

Prospective clinical study. Dogs were categorized according to disease severity as ACVIM Stage B1, B2, or C. Seven echocardiographic indices of RV systolic function were measured. Groups were compared by 1‐way ANOVA and Tukey's HSD test. Frequencies of cases with cardiac remodeling falling outside previously established reference intervals were compared using Fisher's exact test. Intra‐ and interobserver measurement variability was calculated for each RV function index.

Results

The indices TAPSE (P = 0.029), RV St_L (P = 0.012), and RV StR_L (P = 0.041) were significantly different between groups. A greater proportion of B2 dogs (7 of 12) had TAPSE values above reference intervals compared with B1 (2 of 12) or C (2 of 12) dogs (P = 0.027). Measurement variability of TAPSE, RV S', and RV St_G was clinically acceptable.

Conclusions and Clinical Importance

Right ventricular systolic function differs between stages of MMVD, increasing in stage B2, and declining in stage C. The prognostic importance of RV function indices, particularly TAPSE, might be worth evaluating in dogs with MMVD.     

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Author Index to Volume 33

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Keyword Index to Volume 26

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Suspected Limbic Encephalitis and Seizure in Cats Associated with Voltage‐Gated Potassium Channel (VGKC) Complex Antibody

Background

Treatment‐resistant complex partial seizures (CPS) with orofacial involvement recently were reported in cats in association with hippocampal pathology. The features had some similarity to those described in humans with limbic encephalitis and voltage‐gated potassium channel (VGKC) complex antibody.

Hypothesis/Objectives

The purpose of this pilot study was to evaluate cats with CPS and orofacial involvement for the presence of VGKC‐complex antibody.

Animals

Client‐owned cats with acute orofacial CPS and control cats were investigated.

Methods

Prospective study. Serum was collected from 14 cats in the acute stage of the disease and compared with 19 controls. VGKC‐complex antibodies were determined by routine immunoprecipitation and by binding to leucine‐rich glioma inactivated 1 (LGI1) and contactin‐associated protein‐like 2 (CASPR2), the 2 main targets of VGKC‐complex antibodies in humans.

Results

Five of the 14 affected cats, but none of the 19 controls, had VGKC‐complex antibody concentrations above the cut‐off concentration (>100 pmol/L) based on control samples and similar to those found in humans. Antibodies in 4 cats were directed against LGI1, and none were directed against CASPR2. Follow‐up sera were available for 5 cats in remission and all antibody concentrations were within the reference range.

Conclusion and Clinical Importance

Our study suggests that an autoimmune limbic encephalitis exists in cats and that VGKC‐complex/LGI1 antibodies may play a role in this disorder, as they are thought to in humans.     

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Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia

Background

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation.

Hypothesis/Objectives

Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect.

Animals

Two affected and 6 clinically normal horses.

Methods

Ex vivo study. Washed platelets were examined for (1) expression of the αIIb‐β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α‐granules; (4) activation of the mammalian target of rapamycin (mTOR)‐protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol‐4‐phosphate‐3‐kinase, class 2B (PIK3C2B) and SH2 containing inositol‐5′‐phosphatase 1 (SHIP1).

Results

Platelets from affected horses expressed normal amounts of αIIb‐β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α‐granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide‐dependent kinase 1 (PDK1) signaling were normal. SH2‐containing inositol‐5''‐phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation.

Conclusions and clinical significance

Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia.     

Effects of recent Leptospira vaccination on whole blood real-time PCR testing in healthy client-owned dogs

Background

Bacterin‐based canine Leptospira vaccines could present a challenge for the use of whole blood real‐time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog.

Objectives

The objective of this study was to assess whether 2 available 4‐serovar vaccines induce a positive real‐time PCR reaction in the blood of healthy recently vaccinated dogs.

Animals

Twenty healthy dogs.

Methods

This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture‐based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real‐time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real‐time PCR tests targeting 2 different genes were performed independently in a blinded fashion.

Results

Both Leptospira vaccines produced positive real‐time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers.

Conclusions and Clinical Importance

Recent vaccination with 2 commercially available vaccines does not interfere with the use of real‐time PCR for the identification of acute Leptospira infection in dogs.     

Noninvasive Clinical Assessment of Systolic Torsional Motions by Two‐Dimensional Speckle‐Tracking Echocardiography in Dogs with Myxomatous Mitral Valve Disease

Background

Left ventricular torsional motion plays an important role for effective pump function. However, noninvasive clinical assessment of torsional deformations by two‐dimensional speckle‐tracking echocardiography (2D‐STE) in dogs with myxomatous mitral valve disease (MMVD) has not been reported.

Hypothesis

Left ventricular torsion is determined by the native orientation of the helical myocardial fibers, such that it might provide better assessment of myocardial function than conventional methods.

Animals

Sixty‐seven client‐owned dogs with MMVD were classified into 3 classes based on the International Small Animal Cardiac Health Council classification and 16 weight‐ and age‐matched healthy dogs.

Methods

Dogs were examined for myocardial deformations by 2D‐STE and were evaluated for peak systolic rotation and rotation rate at each basal and apical view. Dogs also were evaluated for peak systolic torsion and torsion rate.

Results

Peak systolic torsion was higher in class II than in class I (P < .001) dogs. Peak systolic torsion was lower in class III than in class II (P = .001) dogs and controls (P = .003).

Conclusions and Clinical Importance

Torsional deformations assessed by 2D‐STE differed among clinical classes of MMVD. Myocardial torsional deformations by 2D‐STE may provide more detailed assessment of contractile function in dogs with MMVD.     

Epidemiology of Intoxications in Italy

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