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Objective: To assess the accuracy of automated readings of urine dipstick results for assessment of glucosuria in dogs and cats, compare visual versus automated readings of urine glucose concentration, and determine the utility of the urine glucose-to-creatinine ratio (UGCR) for quantification of glucosuria., Sample: 310 canine and 279 feline urine samples., Procedures: Glucose concentration was estimated in 271 canine and 254 feline urine samples by visual assessment of urine dipstick results and with an automated dipstick reader. Absolute urine glucose and creatinine concentrations were measured in 39 canine and 25 feline urine samples by colorimetric assay with a clinical chemistry analyzer (reference standard for detection of glucosuria), and UGCRs were determined., Results: Automated assessment of the urine dipsticks yielded accurate results for 163 (60.1%) canine urine samples and 234 (92.1%) feline urine samples. Sensitivity of the automated dipstick reader for detection of glucosuria was 23% for canine samples and 68% for feline samples; specificity was 99% and 98%, respectively. Visual readings were more accurate than automated readings for both canine and feline urine. The UGCR was significantly correlated with absolute urine glucose concentration for both dogs and cats, yet there was incomplete distinction between dipstick categories for glucose concentration and UGCR., Conclusions and Clinical Relevance: Urine dipstick readings for dogs and cats were useful for ruling glucosuria in when the result was positive but not for ruling it out when the result was negative. The evaluated dipsticks were more accurate for detection of glucosuria in cats than in dogs. Visual dipstick readings were more accurate than automated readings. The UGCR did not appear to provide additional useful information.

OBJECTIVE To evaluate effects of blood contamination on dipstick results, specific gravity (SG), and urine protein-to-urine creatinine ratio (UPCR) for urine samples from dogs and cats. SAMPLE Urine samples collected from 279 dogs and 120 cats. PROCEDURES Urine pools were made for each species (dogs [n = 60] and cats [30]). Blood was added to an aliquot of a pool, and serial dilutions were prepared with the remaining urine. Color and dipstick variables were recorded, and SG and UPCR were measured. For cats, 1 set of pools was used; for dogs, 2 sets were used. Comparisons were made between undiluted urine and spiked urine samples for individual colors. Repeated-measures ANOVA on ranks was used to compare dipstick scores and UPCR results; χ 2 tests were used to compare proteinuria categorizations (nonproteinuric, borderline, or proteinuric). RESULTS Any blood in the urine resulted in significantly increased dipstick scores for blood. In both species, scores for bilirubin and ketones, pH, and SG were affected by visible blood contamination. No significant difference for the dipstick protein reagent results was evident until a sample was visibly hematuric. The UPCR was significantly increased in dark yellow samples of both species. Proteinuria categorizations differed significantly between undiluted urine and urine of all colors, except light yellow. CONCLUSIONS AND CLINICAL RELEVANCE Any degree of blood contamination affected results of dipstick analysis. Effects depended on urine color and the variable measured. Microscopic blood contamination may affect the UPCR; thus, blood contamination may be a differential diagnosis for proteinuria in yellow urine samples.

Background: Dirofilaria immitis infection occurs in dogs and cats, both of which species are clinically affected by mature adult infections. Cats are uniquely affected by immature-adult infections with an inflammatory pulmonary disease called Heartworm-Associated Respiratory Disease (HARD). D. immitis infection causes pulmonary parenchymal and vascular pathology in the dog and cat. Dogs develop pulmonary hypertension and cor pulmonale, whereas the development of pulmonary hypertension is rare in the cat. D. immitis infection in the dog causes alteration of the right ventricular (RV) extracellular matrix, including a decrease in myocardial collagen. In this study, the RV myocardial changes of cats infected with adult and immature-adult D. immitis were assessed., Methods: The cardiopulmonary systems of six groups of SPF cats (n = 9-10 per group) were examined 8 or 18 months after infection with L3 D. immitis. Two groups were untreated and allowed to develop adult HW; two groups were treated with ivermectin starting 3 months post infection, thus allowing HARD but no mature adult heartworms; and two groups were treated with selamectin beginning 1 month post infection, preventing development of L5 or adult heartworms. A group of specific pathogen free (SPF) normal cats was utilized as a negative control (n = 12). Lung pathologic lesions were objectively assessed, and both RV and left ventricular (LV) weights were obtained to calculate an RV/LV ratio. Intramural RV myocardial collagen content was quantitatively assessed., Results: RV/LV weight ratios were not different between groups. Negative control cats had significantly greater RV collagen content than all other affected groups (P = 0.032). Analysis of the RV/LV ratios and collagen content revealed no significant relationship (r = 0.03, P = 0.723, respectively). Collagen content had a modest, but significant, negative correlation, however, with both pulmonary vascular pathology (r = -0.25, P = 0.032) as well as the total pulmonary parenchymal and vascular pathology (r = -0.26, P = 0.025)., Conclusions: Cats infected with mature and immature D. immitis did not develop RV hypertrophy but did demonstrate loss of RV myocardial collagen content. The collagen loss was present at 8 and 18 months after infection in all infected cats. This loss of RV myocardial collagen was correlated with the severity of pulmonary parenchymal and vascular pathology.

The decision to purchase an in-office hematology instrument is typically based on the desire to have immediate access to complete blood count (CBC) data for disease diagnosis and follow-up and perhaps add to the financial bottom line of your practice. The decision regarding which in-office hematology instrument to purchase requires comparison of available instruments, how they function and knowledge of their strengths and limitations, what analytes they report, their ease of use, and their initial and continued costs. Other considerations include instrument space requirements, ability to interact with your existing data management system, the methods used by analyzers, and data accuracy.

An EDTA-anticoagulated blood sample collected from a 1.5-year-old, intact male, English Bulldog was submitted for a CBC. The CBC data and blood smear evaluation revealed borderline high hematocrit (54%, reference interval 37-55%), inappropriate rubricytosis, moderate leukopenia due to both mature neutropenia and lymphopenia, and mild thrombocytopenia. Numerous leukocytes showed evidence of karyolysis, pyknosis, and karyorhexis, and apoptotic bodies were frequent in the background. Many neutrophils had botryoid nuclei characterized by increased numbers of nuclear segments radially arranged with spoke-like, delicate chromatin filaments connecting the segments centrally. The finding of botryoid nuclei and inappropriate rubricytosis was indicative of severe hyperthermia, such as heatstroke. The dog had been exercised a long time during conditions of high temperature and humidity until he collapsed. The dog was diagnosed with severe heatstroke, hypovolemic shock, disseminated intravascular coagulation, and multiorgan dysfunction syndrome. Despite aggressive treatment, the patient died of cardiopulmonary arrest. Botryoid nuclei are frequent in people with heatstroke. In the authors' experience, botryoid nuclei are seen commonly in dogs with heatstroke, but they have never been reported in veterinary medicine. The presence of petechiation with only mild thrombocytopenia and inappropriate rubricytosis also is suggestive of heatstroke and manifests ongoing life-threatening vascular derangement., (© 2013 American Society for Veterinary Clinical Pathology.)

Background: This study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas., Methods: Dog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs., Results: Overall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC., Conclusions: The results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.

To have an in-clinic hematology instrument in your practice and how it is used are decisions that precede the purchase of an instrument. Advantages and limitations of the various instruments should be considered. Initial purchase cost, reagent/disposable costs, costs of training personnel in the use and care of the instrument, and service/repair contract costs need to be considered. Once the decision is made to have an in-office hematology instrument in your practice you should benefit from having nearly immediate CBC data results that enable you to provide better quality medicine, more rapid clinical decisions, more closely monitor patients for complications of disease or response to treatment. It should also generate revenue and allow some of your staff members to expand and develop their technical skills as they learn the nuances of a new diagnostic tool and how to provide you with the most accurate CBC information. In the final assessment, the addition of an in-office hematology instrument should improve the quality and efficiency of the medical care you provide patients and generate additional practice income.

The Bayer Multistix are commonly used for detection and estimation of feline glucosuria by veterinarians and cat owners. A newer product, the Purina Glucotest, utilizes the same enzymatic technology for detection of glucose, but has been designed for home use as a litter additive that allows interpretation of glucosuria over an 8-h period. The objectives of this study were to assess the sensitivity, specificity, and accuracy of the Glucotest and Multistix, and to assess the 8-h color stability of the Glucotest. Overall, the Glucotest had greater sensitivity and specificity than the Multistix, and more accurately estimated urine glucose concentration if evaluated at least 30 min after exposure to urine. A significant lack of agreement between the results obtained immediately after exposure to urine vs after 30 min and 8 h contradicts the 8-h color stability claim, but the change in urine glucose concentration estimation over time resulted in improved test accuracy at the 30 and 480 min time points., (Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study was to assess safety and alterations in body fluid concentrations of voriconazole in normal horses on days 7 and 14 following once daily dose of 4 mg/kg of voriconazole orally for 14 days. Body fluid drug concentrations were determined by the use of high performance liquid chromatography (HPLC). On day 7, mean voriconazole concentrations of plasma, peritoneal, synovial and cerebrospinal fluids, aqueous humor, epithelial lining fluid (ELF), and urine were 1.47 +/- 0.63, 0.61 +/- 0.22, 0.70 +/- 0.20, 0.62 +/- 0.26, 0.55 +/- 0.32, 79.45 +/- 69.4, and 1.83 +/- 0.44 microg/mL respectively. Mean voriconazole concentrations in the plasma, peritoneal, synovial and cerebrospinal fluids, aqueous humor, ELF and urine on day 14 were 1.60 +/- 0.37, 1.02 +/- 0.27, 0.86 +/- 0.25, 0.64 +/- 0.21, 0.68 +/- 0.13, 47.76 +/- 45.4 and 3.34 +/- 2.17 respectively. Voriconazole concentrations in the bronchoalveolar cell pellet were below the limit of detection. There was no statistically significant difference between voriconazole concentrations of body fluids when comparing days 7 and 14. Results indicated that voriconazole distributes widely into body fluids.

The objective of this study was to investigate the effect of the degree and duration of early dietary AA restrictions on subsequent and overall pig performance and physical and sensory characteristics of pork. For the grower (G) and finisher-1 (F1) phases, 3 corn-soybean meal diets were formulated to contain 100, 80, or 60% of the 1998 NRC total Lys recommendations (100G, 80G, or 60G, and 100F1, 80F1, or 60F1, for the G and F1 phases, respectively). For the finisher-2 (F2) phase, a common corn-soybean meal diet was formulated to satisfy the 1998 NRC total Lys recommendation. Thirty gilts and 30 castrated males (2 gilts or 2 castrated males/pen) were randomly assigned to 5 dietary treatments (100G-100F1, 80G-100F1, 80G-80F1, 60G-100F1, and 60G-60F1) when BW was 22.7 +/- 0.3 kg. Pigs were switched to F1 and F2 diets at 50.7 +/- 0.4 and 79.9 +/- 0.5 kg of BW, respectively. Pigs had ad libitum access to feed and water. All pigs were slaughtered at 110.7 +/- 0.5 kg of BW, and LM samples were collected. Pigs fed the 60G diet had less (P < or = 0.05) ADG during the G phase and greater (P < or = 0.05) ultrasound backfat (UBF) at the end of the G phase than those fed the 100G diet. The ADG decreased linearly (R(2) = 0.70; P < 0.001) as the degree of AA restrictions became more severe. Although serum total protein (TP) and albumin concentrations in pigs fed the 60G-100F1 diets were less (P < or = 0.05) than those fed the 100G-100F1 diets at the end of the G phase, TP concentration was similar between the 2 groups at the end of the F1 phase. Likewise, ADG during the F1 phase and UBF at the end of the F1 phase in pigs fed the 60G-100F1 diets were similar to those fed the 100G-100F1 diets. Feeding the 80G diet resulted in numerically decreased ADG during the G phase, but there was no difference in ADG during the F1 and F2 phases or UBF at the end of F1 and F2 phases between pigs fed the 80G and 100G diets. Overall, pigs fed the 80G-80F1 diets had similar ADG, but less (P < or = 0.05) fat-free lean gain (LG) than those fed the 100G-100F1 diets. These pigs also had less (P < or = 0.05) serum TP and albumin concentrations than pigs fed the 100G-100F1 diets throughout the study. Pigs fed the 60G-60F1 diets had less (P < or = 0.05) overall ADG and G:F and less (P < or = 0.05) LM area and LG than those fed the 100G-100F1 diets. However, they had a greater (P < or = 0.05) subjective marbling score than those fed the 100G-100F1 diets. The results indicated that pigs fed the 80G-80F1 diets may have exhibited compensatory growth in BW gain, but not in terms of lean accretion. Growth performance and carcass traits of pigs fed the 60G-60F1 diets were reduced, indicating that the restriction may have been too severe or too long or both. Early dietary AA restrictions had no clear effect on physical and sensory characteristics of pork.

Case Description: A 7-year-old 509-kg (1,120-lb) Tennessee Walking Horse mare was evaluated because of bilateral mucosanguinous nasal discharge, intermittent right-sided epistaxis, and worsening dyspnea of 9 months' duration., Clinical Findings: Multiple masses in the nasopharynx were detected via endoscopic and radiographic examinations. Cytologic and histologic examinations of biopsy specimens of 1 mass revealed round yeasts with thick nonstaining capsules and occasional narrow-based budding that resembled cryptococcal organisms., Treatment and Outcome: Oral administration of fluconazole and organic ethylenediamine dihydriodide and intermittent intralesional injections with fluconazole, amphotericin B, and formalin resulted in resolution of lesions for a period of 2.5 years. The horse then developed exophthalmos, recurring clinical signs, and extensive nasopharyngeal masses. The masses were surgically debulked via a large frontonasal bone flap, and the horse was treated with IV injections of amphotericin B and long-term oral administration of fluconazole. Clinical signs did not recur in the following 2-year period. A presumptive diagnosis of cryptococcosis was made following cytologic and histologic evaluations of the masses; results of serologic analysis and fungal culture confirmed infection with Cryptococcus neoformans., Clinical Relevance: Cryptococcal infection of the upper respiratory tract in horses has previously been described as a uniformly fatal disease. As this case report illustrates, medical and surgical treatment of sinonasal cryptococcal granulomas in horses may be successful, but the importance of long-term follow-up and the potential for disease recrudescence should be considered. As efficacious antifungal agents become less expensive, their increased use will likely decrease mortality rates in horses with fungal infections.

The objective of this study was to determine the possibility of replacing soybean meal (SBM) in finisher pig diets completely with hydrolyzed feather meal (FM). Forty-five gilts and 45 castrated males (57.8+/-0.8 kg of BW; 3 gilts or 3 castrated males/pen) were randomly assigned to 5 finisher-1 diets with 6 pens per diet. At an average pen BW of 81.0+/-1.4 kg, pigs were offered finisher-2 diets. Corn-SBM, finisher-1 and finisher-2 positive control (PC) diets were formulated to contain 6.1 and 4.7 g of apparent ileal digestible (AID) Lys/kg, respectively. Corn-FM negative control (NC) diets were formulated to be iso-N to the PC diets. The NC diets were supplemented with Lys and Trp (NRC; NC + Lys and Trp) to alleviate apparent deficiencies based on AID AA in FM reported by the 1998 NRC. In addition, the NC diets were supplemented further with the third limiting AA, Thr (40+3AA; NC + Lys, Trp, and Thr), and fourth and fifth limiting AA, His and Ile (40+5AA; NC + Lys, Trp, Thr, His, and Ile), based on the assumption that the apparent ileal digestibility of all indispensable AA in FM is 40%. Pigs had ad libitum access to feed and water. At the end of the study (112.1+/-1.8 kg), blood samples were collected from all pigs before slaughter. As expected, overall ADFI, AID Lys (representing indispensable AA) intake (LysI), ADG, and G:F were greater and BW gain:LysI (G:LysI) was less in pigs fed the PC diet than those fed the NC diet (P<0.001). Pigs fed the NRC diet grew slower than those fed the PC diet (P=0.004), and their G:LysI tended to be less than those fed the PC (P=0.083) or the 40+3AA and 40+5AA diets (P=0.094). Greater fat-free lean percentage (P=0.005), fat-free lean gain (LG; P<0.001), and LG:F (P<0.001) were observed in pigs fed the PC diet compared with those fed the NC diet, but their LG:F or LG:LysI was similar to those fed the NRC diet. However, LG:F (P=0.030) and LG:LysI (P=0.028) were greater in pigs fed the 40+3AA and the 40+5AA diets than those fed the NRC diet. Pigs fed the 40+5AA diet tended to utilize AID Lys more efficiently for LG than those fed the 40+3AA diet (P=0.068). Subjective meat color (P<0.001), firmness (P=0.004), and marbling (P<0.001) scores were greater in pigs fed the 40+5AA diet than those fed the 40+3AA diet. Diets seemed to have no clear effect on the weight of liver, kidney, and heart or serum urea N and triacylglycerides. Pigs fed the PC diet had more serum albumin and total protein (P<0.001), but less glucose (P=0.031) and cholesterol (P<0.001) than those fed the NC diet. Serum total protein was greater (P=0.031) in pigs fed the 40+5AA diet than those fed the 40+3AA diet. The results indicated that the corn-FM diets were not as effective as the corn-SBM diet in supporting BW gain of finisher pigs. However, the results seemed to indicate that pigs fed the corn-FM diet supplemented with the necessary AA can utilize feed and AA for BW gain and lean gain as efficiently as those fed the corn-SBM diet. Further research is needed to explore fully the optimal AA supplementation strategy to effectively utilize FM for pig production.

Background: With more use of bench-top in-office hematology analyzers, the accuracy of reported values is increasingly important. Instruments use varied methods for cell counting and differentiation, and blood smears may not always be examined., Objective: The purpose of this study was to compare canine CBC results using 4 bench-top instruments (Hemavet 950, Heska CBC-Diff, IDEXX LaserCyte, and IDEXX VetAutoread) with ADVIA 120 and manual leukocyte counts., Methods: EDTA-anticoagulated canine blood samples (n=100) were analyzed on each instrument. Manual differentials were based on 100-cell counts. Linear regression, difference plots, paired t-tests, and estimation of diagnostic equivalence were used to analyze results., Results: Correlations of HCT, WBC, and platelet counts were very good to excellent between all in-office instruments and the ADVIA 120, but results varied in accuracy (comparability). Hemavet 950 and Heska CBC-Diff results compared best with ADVIA results and manual leukocyte differentials. HCT and platelet counts on the IDEXX VetAutoread compared well with those from the ADVIA. Except for neutrophil counts, leukocyte differentials from all instruments compared poorly with ADVIA and manual counts. Reticulocyte counts on the LaserCyte and VetAutoread compared poorly with those from the ADVIA., Conclusions: The Hemavet 950 and Heska CBC-Diff performed best of the 4 analyzers we compared. HCT, WBC, and platelet counts on the LaserCyte had minimally sufficient comparability for diagnostic use. Except for neutrophils (granulocytes), leukocyte differential counts were unreliable on all in-office analyzers. Instruments with a 5-part leukocyte differential provided no added benefit over a 3-part differential. Assessment of erythrocyte regeneration on the LaserCyte and VetAutoread was unreliable compared with the ADVIA 120.

The purpose of this study was to determine the immunophenotype and histologic classification of 50 cases of feline gastrointestinal lymphoma. Classification was determined using the National Cancer Institute Working Formulation and the Revised European and American Lymphoma/World Health Organization classification system. Tissue sections were stained with HE, phosphotungstic acid hematoxylin, anti-CD3, anti-CD79a, and anti-BLA.36. Overall, B-cell tumors predominated at 54% (27/50), including 16 diffuse large with immunoblastic nuclear type, 2 diffuse large with centroblastic nuclear type, 3 small lymphocytic, 4 lymphocytic intermediate type, and 2 T-cell-rich large B-cell lymphomas. T-cell tumors comprised 38% (19/50), including 15 epitheliotropic small lymphocytic and 4 lymphoblastic. Three tumors (6%) were nonreactive for B- and T-cell markers and had eosinophilic cytoplasmic granules when stained with HE. Gastric tumors were diagnosed in 24% (12/50) of cats, and 18% (9/50) were present only in the stomach. All gastric lymphomas were of B-cell lineage. Small intestinal lymphoma predominated, with 74% (37/50) of cats affected: T-cell tumors comprised 52% (19/37); 38% (14/37) were B-cell tumors; 8% (3/37) were nonreactive for B- and T-cell markers; and 2% (1/37) expressed both CD3 and BLA.36. Of the 8 cats (16%) that had lymphoma of the large intestine, 88% (7/8) had B-cell tumors and 12% (1/8) had T-cell tumors. The strongest association between gastrointestinal lymphoma immunophenotype, histologic classification, and location occurred in the stomach, where there was a predominance of diffuse large B-cell lymphoma of immunoblastic nuclear type.

Background: Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses., Hypothesis: Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO)., Animals: Six healthy horses and 5 horses with RAO., Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell-free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer., Results: Compared with healthy horses, surfactant from RAO-affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7-62.2 microg/mL BALF versus 172; 111-267 microg/mL BALF, P = .0062) and CSP (20.2; 6.4-48.9 microL/mL BALF versus 155; 94.4-248 microg/mL BALF, P = .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6-5.6% versus 6.6; 4.1-7.6%, P = .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO-affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8-22.8, P = .045)., Conclusions and Clinical Importance: This study demonstrates that surfactant from RAO-affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO-affected horses.

Background: Urine protein: urine creatinine (UP:UC) ratio determined from the quantitative measurement of protein and creatinine in a single urine sample is the best feasible assessment of clinically significant proteinuria in dogs and cats. A dipstick that measures urine protein, urine creatinine, and UP:UC ratio has been used in human medicine and could have application for veterinary practice., Objective: The objective of this study was to compare the Multistix PRO dipstick (Bayer Corporation, Elkhart, IN, USA) to other biochemical methods for determination of urine protein and creatinine, and UP:UC ratio in canine and feline urine., Methods: A complete urinalysis, including sulfosalicylic acid (SSA) precipitation, was performed on urine samples submitted to our laboratory between February and April 2003 from 100 dogs and 49 cats. Urine protein and creatinine concentrations were determined by the Multistix PRO dipstick using a Clinitek 50 analyzer (Bayer) and compared with the results of SSA precipitation and quantitative biochemical analysis. The UP:UC ratios from the dipstick results (calculated by the Clinitek 50 and also manually) were compared with those calculated from quantitative values. Pearson product-moment correlation analysis and diagnostic sensitivity and specificity (using quantitative results as the gold standard) were determined., Results: For both canine and feline urine, protein and creatinine concentrations determined by the Multistix PRO correlated closely with quantitative concentrations for protein (dogs r = .78, P = .0001; cats r = .87, P = .0001) and creatinine (dogs r = .78, P = .0001; cats r = .76, P = .0001). The Multistix PRO was more sensitive and less specific than SSA precipitation for diagnosing clinically significant proteinuria. UP:UC ratios obtained by manual calculation of dipstick results correlated best with quantitative UP:UC ratios in dogs, and had higher specificity but lower sensitivity for the diagnosis of proteinuria. In cats, UP:UC ratios determined by the dipstick method did not correlate (r = -.24, P = .0974) with quantitative values., Conclusions: The Multistix PRO, with manual calculation of UP:UC, may be a good alternative for the diagnosis of clinically significant proteinuria in dogs, but not cats. Dipstick creatinine concentration should be considered as an estimate.

Results from previous in vitro experiments in this laboratory suggested that ethanol may affect selection processes in the thymus. To determine whether ethanol allows escape of potentially autoreactive T-cell clones from negative selection, we fed ethanol to sublethally irradiated, young, adult C57BR mice during the time of thymic and splenic repopulation as a new model of human third trimester fetal alcohol exposure. The mice received a whole-body, sublethal dose (6 Gy) of gamma irradiation at 5 to 6 weeks of age. Feeding of a liquid diet providing 25% of calories as ethanol (EDC) or an isocaloric control liquid diet was begun 3 days after irradiation and was continued for 5 weeks. Each EDC mouse had 2 weight- and age-matched controls, 1 pair-fed (PF), and 1 fed ad libitum (AD LIB). Average blood alcohol concentrations (90 to 440 mg/100 ml) were higher than those reported previously for neonatal mice exposed to ethanol through lactation. At 5 weeks after irradiation, the EDC mice had lower total thymocyte numbers (p < 0.05) and a higher proportion of CD4-CD8-thymocytes than either the PF or AD LIB mice (p < 0.05), which is consistent with findings using in utero models of ethanol exposure. Ethanol exposure also altered the proportion of leukocyte subsets in repopulating spleens. B cells were the most sensitive to the detrimental effects of ethanol and, as a percentage of total nucleated cells in the spleen, B cells were decreased in the EDC group, compared with both the PF and AD LIB groups (p < 0.05). C57BR mice normally delete by negative selection thymocytes bearing v beta 17 T-cell receptors. There was no discernible effect of ethanol exposure during thymic and splenic repopulation on the expression of V beta 17a on thymocytes and splenic T lymphocytes, indicating that ethanol does not affect negative selection.

Objective: To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation., Animals: 5 mature healthy horses., Procedure: Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 microM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody., Results: Released platelet proteins in the presence of beta-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean +/- SEM) 194 +/- 2, 159 +/- 2, 151 +/- 2, 104 +/- 2, and 95 +/- 1 kd. Immunoblots of released platelet proteins had a prominent 180 +/- 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 +/- 1 and 155 +/- 1 kd) were observed., Conclusions: Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets., Clinical Relevance: An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses.

Excessive or nonphysiologic thrombogenesis and fibrinolysis accompanies many diseases. Several specific proteins involved in the physiologic regulation and maintenance of blood in a fluid state are reviewed in this article. Assays for these proteins or evidence of their function (antithrombin III, protein C, protein S, plasminogen/plasmin, tissue plasminogen activator, plasminogen activator inhibitor I, fibrinogen/fibrin degradation products, and thrombin/antithrombin complexes) are described. Principles, general methodology, and application in veterinary medicine are discussed. Although most of the investigative work and knowledge concerning these proteins and assays has been in human beings, their use and application in veterinary medicine is becoming more available in research laboratories at referral centers and some larger commercial veterinary laboratories. Use and interpretation of these assays will help clinicians and researchers better understand pathophysiologic processes occurring in various diseases associated with thrombogenesis and excessive fibrinolysis.

Young (9 to 10 mo) Aberdeen-Angus bulls (n = 5) in excellent body condition and pastured with approximately 35 other bulls of similar age on a farm in southeastern Alabama had fever, harsh pulmonary sounds, increased respiratory rate and variable amounts of hindlimb and scrotal edema. Bulls had mild microcytic, normochromic anemia. Numerous eperythrozoon organisms were identified on blood smears. Indirect hemagglutination inhibition test results for Eperythrozoon suis antibodies were negative either because E. suis antigens do not cross react with cattle eperythrozoon organism antibodies or blood was collected before there was sufficient time for seroconversion. Bulls had swelling of the scrotal wall, soft testes, and poor semen quality, characterized by low progressive motility and a high percentage of spermatozoa with primary and secondary abnormalities. Some of these abnormalities may be attributed to the age of the bulls. However, loss of scrotal thermoregulation was a major cause of testicular degeneration leading to poor semen quality. Other bulls in the herd had fever, increased respiratory rate, and swollen hindlimbs or scrotum. Subsequent to administration of oxytetracycline, parasitemia resolved rapidly, and clinical signs gradually abated. Four of five bulls successfully passed breeding soundness examinations six months after initial clinical illness. The remaining bull failed twice due to the presence of testicular inflammation.

Eperythrozoon organisms were isolated from the blood of a young beef bull with scrotal and hindlimb edema. Young beef bulls, managed under conditions closely mimicking those used in organized bull testing programs, were experimentally administered eperythrozoon organisms. Scant to few organisms were identified on blood smears from bulls (5 of 6) for 2 to 4 days starting 12 days after administration. After a second challenge with intravenously administered viable eperythrozoon organisms, the bulls demonstrated immunity by either failing to become parasitemic (4 of 6) or rapidly clearing the organisms from the blood (2 of 6). No bull became anemic, icteric, or hypoglycemic. Increased serum lactate and decreased blood bicarbonate concentrations probably reflected increased glycolytic activity of infected erythrocytes. A cause for azotemia observed late in the study was not determined. The bulls did not develop scrotal or hindlimb edema. Scrotal circumference and texture remained constant throughout the study. Semen quality was minimally altered while the bulls had organisms identified on blood smears. Harsh lung sounds were asculted in bulls during and immediately after organisms were present in the blood. Although the bulls reliably had organisms in the blood, none showed anemia, scrotal or hindlimb edema, or decreased semen quality; therefore, an additional factor or factors, or greater parasite load may be required for the expression of disease.

Platelets contribute to prethrombotic or thrombotic states; however, accepted evaluation methods (i.e., in vitro testing by use of an aggregometer) of platelet function in cats can be difficult because of the large volume of blood required from which platelets are isolated and the potential for platelet activation due to difficult venipunctures in sometimes uncooperative or excited animals. The activation problem also contributes to errors in platelet counts. Platelets from four domestic short haired cats (two males, two females, 2-3 years old) minimally restrained without sedation or anesthesia were evaluated. Blood (5 ml) was collected by jugular venipuncture directly into syringes containing 3.8% trisodium citrate (nine parts blood to one part anticoagulant) plus prostaglandin E1 (3 microM; 0.25, 0.5, 1, or 2 microliters/500 microliters citrate) or 3.8% trisodium citrate alone. Prostaglandin E1, which is a stable metabolite of arachidonic acid with platelet inhibitory properties similar to those of prostaglandin I2, was added to the anticoagulant to prevent activation of platelets during the collection process. Feline platelets exposed to prostaglandin E1 became immediately and persistently nonreactive to agonists, which negated their use in functional studies (aggregation, 14C-serotonin release, binding of fluorescein-conjugated antifibrinogen) but improved platelet counting accuracy. Detection of in vivo activation of platelets in prethrombotic and thrombotic states in humans has been done by identification of activation-dependent molecules on platelet surfaces by use of specific antibody recognition and detection by flow cytometric analysis. Many activation-dependent platelet surface receptor changes are species specific; however, fibrinogen appears to be conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebrospinal fluid and serum were obtained from 17 adult, healthy llamas (9 males, 1 castrated male, and 7 females). Osmolality; activities of lactate dehydrogenase and creatine kinase; and concentrations of glucose, sodium, chloride, potassium, total protein, and albumin were determined in serum and CSF. Total and differential cell counts were determined in CSF, and electrophoresis of CSF proteins was performed. Total nucleated cell count was low, 0 to 3/microliters, which is lower than that reported for other domestic species and is similar to values in healthy people. Differential leukocyte percentages were disparate depending on the degree of blood contamination. Blood contamination influenced the percentage of neutrophils and eosinophils in CSF. Samples with few erythrocytes had differential leukocyte distribution similar to that of other species: mostly lymphocytes, fewer monocytoid cells, and scant neutrophils. Older llamas had a few eosinophils in the CSF. Total protein, albumin, and gamma-globulin concentrations in llamas were similar to values in cattle and were higher than values in most domestic species. Glucose concentration in CSF was approximately 40% of the value in serum (nonruminant animals and peoply typically have CSF glucose concentration that is approximately 60 to 80% of the serum glucose concentration). Sodium and Cl concentrations in CSF were higher than those in serum, whereas K concentration was lower in CSF, compared with serum. Activities of creatine kinase and lactate dehydrogenase in CSF were markedly lower than those in serum, and the ranges of values in this group of healthy llamas were narrow.

Platelet function, antithrombin and plasminogen activities, and fibrinolytic capabilities in 11 cats with acquired heart disease were compared with results in 4 healthy cats. Of 11 cats with heart disease, 9 had hyperthyroidism with secondary cardiac dysfunction. One cat with hyperthyroidism had renal disease and heart failure, and of 2 cats with idiopathic hypertrophic cardiomyopathy, 1 also had renal disease. At the time of testing, 3 cats had thromboembolic events associated with the disease. Compared with healthy cats, cats with acquired heart disease had increased activity of antithrombin III, a protein that behaves as an acute-phase reactant. Plasminogen activity was decreased, although not significantly, in cats with acquired heart disease, compared with results in healthy cats. In cats with left ventricular dysfunction, clot retraction was decreased (marginal significance, P = 0.058) and might be attributed, in some cases, to the medications received by the cats. Dilute whole blood clots from all cats failed to lyse in vitro. This observation, at present, lacks adequate explanation. Platelets from cats with acquired heart disease, compared with platelets from healthy cats, had decreased responsiveness (aggregation and [14C]serotonin release) to adenosine diphosphate and increased responsiveness to collagen. Hyperthyroid cats were receiving various drugs (propranolol, atenolol, or diltiazem) to empirically treat clinical signs of disease attributable to cardiac dysfunction. Although numbers of cats in each group were small, definite trends were observed in the results of tests. Platelets from cats receiving atenolol had decreased responsiveness to adenosine diphosphate and unaltered responsiveness to collagen, compared with platelets from healthy cats, and may have decreased risk of thrombus formation.(ABSTRACT TRUNCATED AT 250 WORDS)

We characterized the clinicopathologic manifestations of experimentally induced endotoxin-induced mastitis. Responses to hypertonic fluid therapy also were assessed. Eight cows received 1 mg of endotoxin by intramammary infusion in the left forequarter. Four hours after endotoxin administration, cows received 0.9% NaCl, 5 ml/kg of body weight (n = 4) or 7.5% NaCl, 5 ml/kg (n = 4) IV. Endotoxin-infused cows had expanded plasma volume, hyponatremia, transient hyperchloremia and hypophosphatemia, increased serum glucose concentration, and decreased serum activities of liver- and muscle-specific enzymes. Calculated plasma volume increased at 6 hours in cows receiving hypertonic NaCl, and at 12, 24, and 48 hours after endotoxin infusion in both groups. Concurrent observations of decreased serum protein concentration, erythrocyte count, and hematocrit supported observations of increased plasma volume. Relative plasma volume was greater in cows receiving hypertonic NaCl (124.3%) than in cows receiving isotonic NaCl (106.6%) at 6 hours after endotoxin infusion. Cattle receiving hypertonic NaCl had increased voluntary water intake after IV fluid administration. Increased water consumption was not accompanied by increased body weight, indicating probable occurrence of offsetting body water loss. Serum sodium concentration in cows receiving hypertonic NaCl was increased 2 hours after fluid administration, but the magnitude of the change was minimal (< 4 mmol/L) and transient, indicating rapid equilibration with either interstitial or intracellular spaces. Serum sodium concentration was decreased in cows receiving isotonic NaCl at 12, 24, and 48 hours after endotoxin administration, compared with concentration prior to endotoxin administration, indicating selective loss of sodium.

A 16-month-old Charolais bull was examined because of acute onset of scrotal and hind limb edema, fever, tachycardia, tachypnea, anorexia, and lethargy. Scrotal circumference on initial examination was 48 cm. Clinicopathologic abnormalities included microcytic, normochromic anemia and numerous Eperythrozoon organisms in blood smears. Results of immunohistochemical staining of a skin biopsy specimen suggested that the edema was the result of an Arthus-type reaction. Semen quality deteriorated rapidly, and the bull was aspermic within 7 days. The bull was treated with oxytetracycline, and the anemia and edema gradually subsided. Eperythrozoon organisms were not detected in blood smears after 3 days. Six months after initial examination, results of physical examination and semen evaluation were normal. We hypothesize that scrotal edema caused failure of testicular thermoregulation, resulting in transient production of abnormal sperm and infertility.

Anesthesia was induced and maintained in 6 Suffolk wethers by continuous i.v. infusion of guaifenesin (50 mg/ml), ketamine (1 mg/ml), and xylazine (0.1 mg/ml) in 5% dextrose in water (triple drip) to assess the anesthetic and cardiopulmonary effects. All sheep were positioned in right lateral recumbency. Dosages of triple drip used for induction and maintenance of anesthesia were 1.2 +/- 0.02 ml/kg and 2.6 ml/kg/h, respectively. Lack of gross purposeful movement of sheep to electrical stimulation indicated that analgesia and muscular relaxation induced by triple trip were adequate for surgical procedures. Heart rates and arterial blood pressure remained unchanged from baseline values during a 1-hour period of anesthesia. Arterial blood pressures were measured indirectly, using an inflation cuff placed over the metatarsal artery at the heart level. Significant decrease in arterial partial pressure of O2 (PaO2), coupled with an increase in arterial partial pressure of CO2 (PaCO2), from baseline values was observed throughout the course of the study. Decrease in PaO2 was observed concomitantly with significant (P < 0.05) increase in respiration rate. Changes in arterial blood gas tensions observed in this study were attributed to respiratory depressant effect induced by anesthetic drugs and right-to-left shunting, perfusion/ventilation mismatch, or both caused by right lateral recumbency. Administration of 100% O2 via the endotracheal tube reduced the magnitude of the decrease in PaO2. All sheep recovered smoothly and stood within 96.3 +/- 48.9 minutes after termination of triple drip administration.

Eight tests of hemostasis were measured in 233 horses with colic. Blood samples were obtained at admission and for 4 consecutive days of hospitalization. Data were analyzed retrospectively by outcome, by broad-category diagnosis group, by small intestinal disorder, and by smaller categories for comparing specific diseases. Nonsurviving horses and horses with the most severe forms of intestinal ischemia had changes interpreted as hypercoagulative, the intensity of which was increased on the first and second mornings (sample times 2 and 3) after admission, when most significant differences for results of specific tests were detected. Nonsurvivors had decreased antithrombin III activity and prolonged prothrombin and activated partial thromboplastin times; those with strangulating obstructions also had decreased protein C and plasminogen activities. During hospitalization and with survival, these changes tended to reverse. In most horses, regardless of diagnosis or outcome, concentration of fibrin degradation products and fibrinogen, and alpha 2-antiplasmin activity increased over time. Whether these changes reflected specific effects of colic or of the acute-phase response was not determined. In comparisons of small intestinal disorders (proximal enteritis, strangulations, and impactions), diagnostically distinguishing features were not found. Likewise, in comparisons of specific diseases (small vs large intestinal impaction, proximal enteritis vs colitis, small vs large intestinal obstruction), diagnostically distinguishing features were not found.

Cats with cardiomyopathy, especially dilated cardiomyopathy associated with taurine deficiency, often develop systemic thrombi. To investigate the relation of taurine deficiency to formation and persistence of thrombi, cats were made taurine-deficient by consumption of a casein-based taurine-deficient diet, then were evaluated for anticoagulant and profibrinolytic activities and platelet function. The cats served as their own controls in the taurine-replete state; then, values were compared for the taurine-deficient state. Plasma (P < 0.01), blood (P < 0.05), and platelet (P < 0.05) taurine concentrations were decreased markedly after cats consumed the taurine-deficient diet for 6 weeks, compared with baseline concentrations before diet. Compared with the taurine-replete state, taurine deficiency induced significantly (P < 0.05) increased mean antithrombin III activity, no significant change in plasminogen and fibrinolytic activities, and similar clot retraction/lysis test results. Decreased (P < 0.01) adenosine diphosphate (ADP)-induced platelet aggregation and [14C]serotonin release, and slightly increased (P < 0.05) collagen-induced platelet [14C]serotonin release, but unchanged collagen-induced platelet aggregation were observed in taurine-deficient cats, compared with taurine-replete cats. Changes in antithrombin III activity most likely reflected hepatocellular acute-phase reaction, which indicates that taurine deficiency may induce a stress-responsive state. Results of platelet function testing indicate that taurine may modulate platelet responsiveness to physiologic agonists, but not in consistent manner. That platelets from the taurine-deficient cats had decreased responsiveness to ADP, but increased responsiveness to collagen is surprising, because irreversible aggregation is mediated by release of granule-associated ADP after sufficient initial stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemostasis was evaluated in cows with experimentally induced endotoxemia and mastitis, caused by intramammary infusion of endotoxin (1 mg) derived from Escherichia coli. Hemostatic tests included prothrombin time; activated partial thromboplastin time; thrombin time; fibrinogen, fibrin(ogen) degradation products, and platelet concentrations; and antithrombin-III and plasminogen activities. Significant alterations were observed in the mean values of most analytes (prothrombin time was increased; thrombin time was increased with subsequent decrease; activated partial thromboplastin time, fibrinogen concentration, plasminogen activity, and platelet concentration were decreased; and antithrombin-III activity and fibrin(ogen) degradation products concentration were unchanged) at 1 or more postchallenge sample collection times (3, 12, or 24 hours) after endotoxin administration, compared with mean values obtained from samples prior to endotoxin administration. These data indicated activation of hemostatic mechanisms, initiated either directly by endotoxin or by inflammatory mediators released or produced in response to endotoxin infusion.

This study examined the safety of intravenous hypertonic saline in cattle with experimental gram-negative endotoxemia. Cerebrospinal fluid (CSF) composition was examined in five control cows and eight treated cows 24 hours after the intramammary infusion of 1 mg of endotoxin. Four of the endotoxin challenged cows were treated intravenously with isotonic (0.9%) sodium chloride and four cows were treated intravenously with hypertonic (7.5%) sodium chloride. Decreased CSF osmolality, and sodium and alpha globulin concentrations and increased CSF concentrations of beta globulin were observed in both endotoxin-challenged saline-treated groups. No CSF compositional differences were observed between endotoxin-challenged cows receiving isotonic or hypertonic saline. Although no cytologic or biochemical evidence of salt poisoning was observed in cows receiving hypertonic saline, significant changes were observed in the CSF composition of both endotoxin-infused saline-treated groups.

Cerebrospinal fluid and serum were obtained from 16 clinically normal adult cows (11 dairy, 5 beef). Sodium, potassium, magnesium, total protein, and albumin concentrations, osmolality, and lactate dehydrogenase and creatine kinase activities, were quantified in CSF and serum. Total and differential cell counting, protein electrophoresis, and IgG quantification were performed on CSF. Statistical analyses of these variables, including mean, SEM, range, and 95% confidence intervals, were performed. Effects of blood contamination were evaluated, and were found to be negligible for all measured constituents. Correction factors for CSF creatine kinase and lactate dehydrogenase activities accounting for cellular contamination were developed. Total nucleated cell count was similar to counts in CSF of other species, but higher than values in healthy people. Differential leukocyte count in CSF was similar to that reported in CSF of other domestic animals: mostly lymphocytes, fewer monocytoid cells, and scant neutrophils. Cerebrospinal fluid protein concentration was higher than concentration reported for dogs, goats, and people, but was similar to values reported for horses. Beef cows had higher CSF total protein concentration than did dairy cows; also, beef cows had higher CSF gamma-globulin concentration. The concentration of sodium in CSF was slightly higher than the value in serum, and potassium concentration was lower than the value in serum. In contrast to studies of human beings, CSF osmolality was generally less than serum osmolality in the cows studied. Reference values for CSF electrolyte concentrations and osmolality are useful for diagnosis of salt poisoning and for assessment of the effects of fluid therapy. Magnesium concentration was lower in CSF, compared with serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein C content and plasminogen activity were measured in plasma from 100 horses with signs of colic. Data were analyzed by grouping horses 4 ways. Each horse was allotted to 1 of 2 outcome groups (survivors and nonsurvivors), 1 of 3 broad-category diagnosis groups (inflammatory disorders, strangulating obstructions, and all other gastrointestinal disorders), and 1 of 2 clinical management groups (medical and surgical). In a fourth grouping, all horses (although numbers of horses included in each subgroup were small) were assigned either to specific diagnostic groups that had high expectation for activated hemostasis (intestinal ischemia, endotoxemia, jugular thrombosis, peritoneal adhesions, and laminitis) or to a control group, in which active hemostasis was unlikely. Within 2 to 24 hours after admission, nonsurvivors developed lower protein C content than did survivors. Protein C content and plasminogen activity became low during hospitalization in horses with strangulating obstructions and in horses having surgery. The results from the grouping by specific diagnosis must be considered pilot data because the numbers of horses in each subgroup were small. Although not statistically significant, trends were noticed in protein C and plasminogen: (1) horses with intestinal ischemia and endotoxemia developed low protein C content and plasminogen activity, (2) protein C content became low in horses that developed peritoneal adhesions or laminitis, and (3) plasminogen activity became low in horses that developed jugular thrombosis. Low protein C content or low plasminogen activity, or both, may be useful as predictors for outcome and for these specific complications of equine colic.(ABSTRACT TRUNCATED AT 250 WORDS)

An antigenic assay was developed for determination of protein C in horses. Protein C, a natural, vitamin K-dependent anticoagulant component in blood, was isolated from equine plasma, a specific antibody was produced in goats, and a rocket electroimmunophoresis assay was established. Tests were performed to verify the identity of the isolated protein C and to determine the purity of the antibody. Protein C antigen was measured in plasma from 34 clinically normal horses, and values were compared with amidolytic function values. The mean (+/- SD) values for the 2 test methods were similar (antigen content, 104.5 +/- 13.8%; amidolytic activity, 104.6 +/- 17.5%), but the correlation coefficient was 0.1. Four horses given Na coumarin had markedly decreased plasma protein C amidolytic activity and minimal decrease in protein C antigen content.

A functional assay for equine plasminogen was established, using urokinase as the activator, a synthetic chromogenic substrate, a computer-assisted centrifugal analyzer, and acidified/neutralized plasma. One documented effect of plasma acidification appears to be inactivation of alpha-2-antiplasmin. Intra- and interassay precision testing yielded coefficients of variation of 4.1% (n = 10) and 5.6% (n = 26), respectively. Plasminogen was stable in equine plasma stored up to 1 week at 4 C and up to 5 months at -70 C. Plasminogen in nonacidified equine plasma was not activated by urokinase, streptokinase, tissue plasminogen activator, or tissue plasminogen activator plus soluble fibrin. Streptokinase also failed to activate plasminogen in acidified/neutralized equine plasma.