Effects of hemolysis on serum chemistry analytes in ratites

Since hemolysis has been a common problem in submitted ratite serum samples, a study was performed to determine interference by hemolysates. Nine chemistry analytes including glucose, total protein, albumin, creatine kinase (CK), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AS7), calcium (Ca), phosphorus, and uric acid were evaluated on a wet reagent analyzer (Ciba Corning Express 550) and on a dry slide reagent analyzer (VetTest 8008). In emus, increasing hemoglobin concentrations increased total protein, albumin, and CK for both analyzers. With increasing hemoglobin concentrations, the Ciba Corning 550 analyte values were increased for AST, Ca, and uric acid and decreased for glucose and phosphorus, the opposite effect was seen in values from the VetTest 8008. GGT levels were variable or sometimes undetectable. Changes in ostrich analytes with hemolysis were similar to emus using the same analyzer. The effects of serum hemolysis often differed in magnitude and direction between the two chemistry analyzers. Interferographs were constructed to aid in rapid assessment of the effects of hemolysis on submitted serum samples.     

Effects of hemolysis, lipemia, hyperbilirrubinemia, and anticoagulants in canine C-reactive protein, serum amyloid A, and ceruloplasmin assays

The objective of the present study was to determine the effects that hemolysis, lipemia, bilirubinemia, and anticoagulants might have on the most commonly used assays for C-reactive protein and serum amyloid A, and determination of ceruloplasmin values in dogs. Solutions of hemoglobin, lipid, and bilirubin were added to serum aliquots. Additionally, serum and plasma samples with different anticoagulants (heparin, EDTA, and citrate) were obtained from healthy dogs. Hemolysis, lipemia, and hyperbilirubinemia interfered significantly with the C-reactive protein and ceruloplasmin results, but not with those for the serum amyloid A assay. The use of anticoagulants produced significant changes in the results for the assays tested. However, the magnitude of the differences caused by the interfering substances does not appear to have an important impact on the clinical interpretation of the tests.     

Angiostatin and integrin alphavbeta3 in the feline, bovine, canine, equine, porcine and murine retina and cornea

PURPOSE: Angiogenesis is tightly controlled in the ocular tissues of domestic animals but its mechanisms are not fully understood. This is largely because of insufficient data on the expression of molecules that impact angiogenesis. Because angiostatin and one of its receptors integrin alphavbeta3 inhibit and promote angiogenesis, respectively, we hypothesized that the normal retina and cornea of domestic animals would express angiostatin but not integrin alphavbeta3. PROCEDURE: Normal eyes of the cat, cow, dog, horse, pig and rat were evaluated for angiostatin and integrin alphavbeta3 by light and electron immunocytochemistry and estern blots. RESULTS: Angiostatin was detected in the corneal epithelium of the cat, dog, horse, pig and rat, but was not found in cow corneal epithelium. Angiostatin was localized in the nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and the photoreceptor layer of the cat, cow, dog and rat. Horse and pig retinas showed additional staining in the matrix of the inner nuclear layer. Immunogold electron microscopy further confirmed angiostatin in cat retina. Western blots showed angiostatin in corneal and retinal homogenates. Integrin alphavbeta3 was absent in cornea and retina of all the species studied. CONCLUSION: These data show that angiostatin, an inhibitor of angiogenesis, is present while integrin alphavbeta3, which promotes angiogenesis, is absent in normal cornea and retina of the domestic animals in this study with the exception being angiostatin absence in cow corneal epithelium. Therefore, angiostatin may contribute to the anti-angiogenic environment in the normal domestic animal eye while its absence in the cow may contribute to greater propensity for corneal vascularization. Because integrin alphavbeta3 is one of the receptors for angiostatin, its absence may prevent angiostatin from killing normal retinal and corneal cells.     

Ultrastructure of canine, feline, and bovine mast cell neoplasms

Biopsy specimens were collected from skin or spleen from 8 dogs, 7 cats, and 1 cow with mast cell neoplasms. Following histopathologic grading, the neoplasms were examined by transmission electron microscopy with the intention of reviewing their fine structure and recording new findings. For 2 feline specimens, short-term cell cultures were established, and adherent cells were fixed in situ and examined with the electron microscope. In addition to the usual array of mast cell organelles, including Golgi apparatus, secretory granules, mitochondria, vesicles, tubules, microfilaments, and ribosomes, important findings included coarse interdigitation of cytoplasmic projections between adjacent mast cells; intracytoplasmic parallel stacks of 10 nm diameter filaments as well as parallel arrays of coarse, 120 to 150 nm diameter tubules; the appearance of coated vesicles and caveolae on cell surfaces; the appearance of endocytosed erythrocytes; and the formation of giant secretory granules.     

Analysis of feline,canine and equine hemograms using the QBC VetAutoread

Blood samples form 120 consecutive clinical cases (40 cats, 40 dogs and 40 horses) were analyzed on the QBC VetAutoread analyzer and the results compared with those obtained by a Baker 9000 electronic resistance cell counter and a 100-cell manual differential leukocyte (WBC) count. Packed cell volume (PCV), hemoglobin (Hb) concentration, mean cell hemoglobin concentration (MCHC), and platelet, total WBC, granulocytes, and lymphocyte plus monocyte (L+M) counts were determined. Indistinct separation of red blood cell and granulocytes layers on the QBC VetAutoread was observed in samples from five cats (12.5%), two dogs (5%), and one horse. Significantly different (P=0.002) median values for the two methods were obtained for PCV, Hb concentration, MCHC and platelet count in cats; PCV, MCHC, WBC, count and granulocytes count in dogs; and PCV, Hb concentration, MCHC and WBC, granulocytes and platelet counts in horses. Results from the QBC VetAutoread should not be interpreted using reference ranges established using other equipment. Results were abnormal on a limited number of samples; however, when correlation coefficients were low, marked discrepancy existed between values within as well as outside of reference ranges. Spearman rank correlation coefficients were excellent (r=0.93) for PCV and Hb concentration in dogs, and Hb concentration and WBC count in horses. Correlation was good (r=0.80-0.92) for PCV and Hb concentration in cats, WBC count in dogs, and PCV, granulocytes count and platelet count in horses. For remaining parameters, correlation was fair to poor (r=0.79). Acceptable correlations (r>0.80) were achieved between the two test systems for all equine values except MCHC and L+M count, but only for PCV and HB concentration in feline and canine blood samples.     

Stability of sorbitol dehydrogenase activity in bovine and equine sera

Serum sorbitol dehydrogenase (SDH) activities in 10 cows and nine horses were measured using an automated clinical analyzer. The serum samples were divided into aliquots that were stored at room temperature (21 degrees C), refrigerated (0-5 degrees C), or frozen (-30 degrees C). The stability of the SDH activity was monitored at various intervals. SDH activity in bovine sera remained stable for at least 5 hours at room temperature, 24 hours refrigerated, and 72 hours frozen without any significant (p < 0.05) differences from the initial serum values. In equine sera, SDH activity remained stable for at least 5 hours at room temperature and 48 hours frozen. The activity of the refrigerated equine sera was stable for at least 5 hours but less than 24 hours. An evaluation of fresh bovine serum and heparinized plasma samples indicated that there was no significant difference (p < 0.05) between the two sampling methods and that either may be employed for automated measurement of SDH activity following the established protocol. Sample type comparison indicated that there was a small but statistically significant (p < 0.05) difference between the results obtained comparing fresh serum and heparinized plasma samples for the horse. A reference range for Holstein cows was established using sera from 71 clinically healthy cattle (mean -/+ 2 SD = 32 -/+ 26 U/L).     

Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine,porcine and feline sera

Monospecific antisera were prepared in rabbits against canine coronavirus (CCV) and transmissible gastroenteritis virus of pigs (TGEV), and in 24 pigs and 3 cats against TGEV alone. Neutralizing antibody titres were higher for the immunizing than the heterologous virus, although cross-neutralization usually was detected. This confirmed that CCV and TGEV are distinct, but antigenically related coronaviruses. In sera from 41 dogs, CCV-neutralizing titres were on average 2.7 fold higher than TGEV-neutralizing titres, suggesting that CCV was the causal agent. Sera from 29 cats in colonies with feline infectious peritonitis (FIP) and known to contain TGEV-neutralizing antibody, were found to have titres 12.3 fold higher against CCV. The FIP virus (FIPV) is probably more closely related to CCV than TGEV as judged by antigens involved in virus neutralization.Antisera to two isolates of bovine coronavirus, three isolates of haemagglutinating encephalomyelitis virus, seven strains of avian infectious bronchitis virus and the 229E strain of human coronavirus all failed to neutralize CCV and TGEV. Thus CCV, TGEV and probably FIPV fall into a group of antigenically related agents, separable from other members of the family Coronaviridae, by both virus neutralization and immunofluorescence tests.     

Validation of a rapid solid-phase radioimmunoassay for canine, bovine, and equine insulin

A rapid and convenient commercial radioimmunoassay kit, developed for quantifying hormones in specimens from human beings, was validated for use in measuring insulin in serum of dogs, cattle, and horses. The procedure uses polypropylene assay tubes treated with rabbit anti-porcine insulin serum and porcine [125I]iodoinsulin. Specificity was proven by demonstrating that standard solutions of porcine insulin and serial dilutions of canine, bovine, and equine sera and pancreatic extracts inhibited binding of [125I]iodoinsulin to the antibody in a parallel manner. Gel-filtration chromatography of pancreatic extracts yielded a major peak of immunoreactive material that eluted identically with [125I]iodoinsulin. Immunoreactivity was not associated with fractions that contain larger and smaller molecular weight peptides (eg, proinsulin and C-peptide, respectively). Biological specificity of the assay was shown by demonstrating increased insulin in serum after injection of glucose into heifers and glucagon into dogs and horses. Purified insulin and insulin in pancreatic extracts could be quantitatively recovered from serum, thereby demonstrating accuracy of the assay. Interassay precision of 5 control specimens run in 20 consecutive assays ranged from 6.7% to 20.1% (coefficient of variation) and intra-assay precision of 6 control specimens each assayed 10 times ranged from 4.4% to 10.7% (coefficient of variation). Sensitivity of the assay was 3.2 microIU/ml. This radioimmunoassay for insulin is ideal for veterinary research and diagnosis, because a single set of reagents and procedures can be used for at least 3 species.     

Binding of 125I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using 125I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.     

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

OBJECTIVE: To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. SAMPLE POPULATION: Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. PROCEDURE: Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. RRESULTS: Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.     

Effects of asphyxia and potassium on canine and feline electrocardiograms.

The effects of asphyxia and potassium on the electrocardiogram (ECG), lead II, were recorded from dogs and cats anesthetized with sodium pentobarbital and halothane. Electrocardiographic recordings were made during control periods, during asphyxia (occluded endotracheal tube), during infusion of an isotonic KCl solution and during infusion of an isotonic NaCl solution. Arterial and venous blood gas partial pressures (PaCO2, PvCO2, PaO2 and and PvO2), plasma Na+ and K+ concentrations, heart rate and mean arterial blood pressure were measured during control periods, asphyxia and during the periods of infusion. The vagi were severed to assess the effect of vagal tone on the ECG changes. The characteristic ECG changes during asphyxia and the electrolyte imbalances resulting from infusion of isotonic KCl and NaCl were determined during sodium pentobarbital and halothane anesthesia in both dogs and cats. The combination of halothane and high PCO2 caused cardiac arrhythmias. Spontaneous recovery from ventricular fibrillation, as a result of hyperkalemia, was recorded from cats. Disappearance of the P waves, which is characteristic of hyperkalemia, was infrequent in this study and the U waves associated with hypokalemia were not found. Severing the vagi did not alter the ECG changes characteristic of asphyxia, hyperkalemia and hypokalemia. It was found that asphyxia and infusion of fluids high or low in potassium can produce ECG changes in both dogs and cats that can be correlated with blood gas partial pressure changes or plasma potassium concentrations.     

A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide

Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.     

Effects of surgical sterilization on canine and feline health and on society

Surgical sterilization of dogs and cats is a well-accepted measure for population control in some countries, but is considered unethical as an elective surgery in other countries. This is a review of what is known regarding positive and negative effects of gonadectomy surgery on individual animals and on societal management of unowned dog and cat populations.     

The distributions of phytohemagglutinin-P and concanavalin A binding sites on equine, bovine and canine peripheral blood lymphocytes

The distributions of phytohemagglutinin-P (PHA) and concanavalin A (ConA) binding sites were investigated for equine, bovine and canine peripheral blood lymphocytes (PBL). Non-B lymphocytes were collected from each PBL using a fluorescence-activated cell sorter (FACS), and the numbers of PHA and ConA binding sites on their surfaces were counted. Most PHA binding sites on PBL of the three species were shown on the surfaces of non-B lymphocytes. On the other hand, the ConA binding sites on equine and canine PBL existed mainly on the surfaces of non-B lymphocytes, but B lymphocytes of these two species had many ConA binding sites. These results were confirmed by the results of two-parameter fluorescence analysis using FACS. It is, therefore, concluded that the different optimum concentrations of PHA and ConA in PBL blastogenic responses of each animal depended on the different distributions of their binding sites.     

Effects of bovine lactoferrin on in vitro replication of feline herpesvirus

Objective To investigate the effects of bovine lactoferrin on in vitro replication of feline herpes virus (FHV‐1) and to determine at what points during viral replication these effects occur. Sample population Cultured Crandell‐Reese feline kidney (CRFK) cells and FHV‐1 strain 727. Procedure Five concentrations of bovine lactoferrin (0.5, 1, 2, 5, and 10 mg/mL) were added at one or more of three time points during conventional plaque reduction assays: (a) uninfected CRFK cells were incubated in lactoferrin‐containing medium for 30 min prior to viral adsorption; (b) virus was suspended in lactoferrin‐containing medium prior to and during adsorption, or (c) CRFK cells were incubated with lactoferrin‐containing medium for 48 h following viral adsorption. Plaques were counted and antiviral effect expressed as percent inhibition relative to control medium that contained no lactoferrin. Results Exposure of CRFK cells to lactoferrin prior to or during viral adsorption inhibited FHV‐1 replication by 87–96% (mean: 91%). Application of lactoferrin following viral adsorption had no appreciable effect on FHV‐1 replication. No additive or synergistic effects were noted when lactoferrin was added at multiple steps. These effects were similar at all concentrations of lactoferrin tested. Cytotoxic effects of lactoferrin on CRFK cells were not observed at any concentration tested. Conclusions and clinical relevance Bovine lactoferrin has a notable inhibitory effect on the in vitro replication of FHV‐1 prior to and during, but not following viral adsorption. These findings strongly suggest that lactoferrin inhibits FHV‐1 adsorption to the cell surface and/or penetration of the virus into the cell. Clinical effects of topical lactoferrin in acute or recrudescent herpetic episodes in cats warrant investigation.