Establishment of the European College of Veterinary Clinical Pathology (ECVCP) and the current status of veterinary clinical pathology in Europe.

After 5 years of development, the European College of Veterinary Clinical Pathology (ECVCP) was formally recognized and approved on July 4, 2007 by the European Board of Veterinary Specialisation (EBVS), the European regulatory body that oversees specialization in veterinary medicine and which has approved 23 colleges. The objectives, committees, basis for membership, constitution, bylaws, information brochure and certifying examination of the ECVCP have remained unchanged during this time except as directed by EBVS. The ECVCP declared full functionality based on the following criteria: 1) a critical mass of 65 members: 15 original diplomates approved by the EBVS to establish the ECVCP, 37 de facto diplomates, 7 diplomates certified by examination, and 5 elected honorary members; 2) the development and certification of training programs, laboratories, and qualified supervisors for residents; currently there are 18 resident training programs in Europe; 3) administration of 3 annual board-certifying examinations thus far, with an overall pass rate of 70%; 4) European consensus criteria for assessing the continuing education of specialists every 5 years; 5) organization of 8 annual scientific congresses and a joint journal (with the American Society for Veterinary Clinical Pathology) for communication of scientific research and information; the College also maintains a website, a joint listserv, and a newsletter; 6) collaboration in training and continuing education with relevant colleges in medicine and pathology; 7) development and strict adherence to a constitution and bylaws compliant with the EBVS; and 8) demonstration of compelling rationale, supporting data, and the support of members and other colleges for independence as a specialty college. Formal EBVS recognition of ECVCP as the regulatory body for the science and practice of veterinary clinical pathology in Europe will facilitate growth and development of the discipline and compliance of academic, commercial diagnostic, and industry laboratories in veterinary clinical pathology. Future needs are in developing sponsorship for resident positions, increasing employment opportunities, increasing compliance with laboratory, training, and continuing education standards, and advancing relevant science and technology.

Distribution and morphology of skeletal muscle lesions after experimental restraint stress in normal and stress-susceptible pigs.

Six normal and 22 stress-susceptible (SS) pigs were subjected to experimental restraint stress to test the hypothesis that SS pigs are more affected by stress-induced skeletal muscle lesions than normal pigs. The stress was provoked by a 12 min intravenous infusion of the myorelaxant succinylcholine at a dose which induced leg paralysis. At necropsy 2-3 days after the stress, 24 muscles were examined macro- and microscopically. The plasma levels of noradrenaline and adrenaline increased significantly during the stress, especially in SS pigs. Significantly higher scores of acute muscle lesions (degeneration and phagocytosis) were recorded in the SS pigs than in the normal pigs. The antebrachial flexor muscles, m. gastrocnemius, crural flexor muscles, m. serratus and m. intercostalis were most affected, while m. semitendinosus, m. masseter, crural extensor muscles, m. quadriceps and antebrachial extensor muscles were the least affected. The muscle regeneration of SS pigs was greater than that of normal pigs indicating more active rhabdomyolysis in SS pigs than in normal pigs. The muscle lesions were also reflected in increased serum levels of creatine kinase (CK), aspartate aminotransferase (ASAT), potassium and creatinine in SS pigs. It is concluded that the restraint stress induced skeletal muscle lesions and increased sympathetic activity, predominantly in SS pigs. It was also shown that certain skeletal muscles are more affected by rhabdomyolysis than others.

Serum auto antibodies and clinical/pathological features in German shepherd dogs with a lupuslike syndrome.

This study presents 8 dogs of German Shepherd breed (6 males, 2 females, 2-5 years of age at onset of the disease) with a lupus like syndrome characterized by febrile polyarthritis, wasting, nephropathy, cutaneous lesions and high positive titres of ANA (antinuclear antibodies) of speckled type. The serum autoantibodies were further characterized by double immunodiffusion against ENA (extractable nuclear antigen), ELISA for Histone antibodies (Histon fraction H-24A and H-3S), indirect IF on rat-liver sections, non treated and RNase/DNase digested sections for DNP/RNP antibodies, and smears of a hemoflagellate C. luciliae for antibodies vs doubbel strained DNA, (dsDNA). Thus, the high ANA titres in these dogs represent varying types of autoantibodies against nucleoproteins of both DNA and RNA nature, associated histone antigens and non-histone antibodies (RNA and Sm) as well. Rheumatoid Factor titres in serum from these dogs were low or negative. Immunoglobulin deposits at dermo-epidermal junctions were demonstrated in some of the dogs with hyperkeratotic skin lesions. High concentration of serum-IgG was a constant finding in combination with anemia and in most cases leukopenia probably related to the chronic inflammatory process in these animals. Autoimmune hemolytic anemia (AIHA) or thrombocytopenia was not detected in these dogs.

Serum creatine kinase activity as a selection criterion for stress susceptibility after standardised stress in pigs.

Estimation of serum creatine kinase isoenzyme activity was used as a selection criterion for stress sensitivity in pigs 8-12 weeks of age, classified for stress susceptibility as positive to halothane anaesthesia. Standardized stress was provoked by administration of adrenocorticotropic hormone or synstigmine. The experiments were performed under experimental conditions in permanently catheterized animals and in a field trial. It was shown that the stress inductors can be used as a selection criterion for stress sensitivity in catheterized animals by estimation of the CK-MM isoenzyme activity 20-24 h after administration of the inductor. In cases where the individual reaction to halothane anaesthesia is unclear and non classifiable, CK-MM isoenzyme assay performed before and 24 h after the halothane test, may also be useful under practical conditions as an additional indicator of stress sensitivity.

A comparative study of different methods for measurement of rheumatoid factor in dog serum.

An enzyme linked immunosorbent assay (ELISA) technique was developed and compared with two routinely used methods, latex agglutination and Rose-Waaler hemagglutination, for the measurement of Rheumatoid Factor (RF) in dog serum. The frequency of positive samples for IgM-RF was rather low, about 8-12% of the 460 dogs with different rheumatic disorders being classified as positive by these methods. It was further concluded that the latex agglutination test used in this study (Synbiotics CRF Kit) should be performed only on heat inactivated serum (56 degrees C for 30 minutes) thereby significantly reducing the frequency of weak, probably false, positive agglutination reactions. When this was considered, a significant correlation was obtained for the three different techniques for RF estimation in serum from normal control as well as rheumatic diseased dogs.

Chemiluminescence and chemotaxis assay of porcine polymorphonuclear cells. A methodological study.

A Boyden-Chamber technique for leukocyte migration was adapted to a polymorphonuclear (PMN) cell concentration of 5 x 10(6) cells/ml an optimal amount of cells for the leading front technique. Casein (0.1%) is so far the most effective chemotactic substance for porcine granulocytes. A formylpeptide++, FMLP (in concentrations of 10(-2) to 10(-10) mmol/l), does not stimulate migration in porcine granulocytes when compared to random migration. For the chemiluminescence assay a concentration of 5 x 10(6) PMN cells/ml buffer is needed to obtain optimal conditions in pig granulocytes. Moreover the chemiluminescence should be performed within a standardized interval after the isolation of leukocytes since the cell activity declines rapidly after the isolation. The chemiluminescence and migration assays were standardized at 37 degrees C; the activity of the granulocytes did not show any significant variation within a temperature interval of 37-39 degrees C, whereas temperatures below 37 degrees C significantly reduce the granulocytic activity.

Chemiluminescence and chemotaxis assay of canine granulocytes: a methodological study.

Chemiluminescence (CL) of isolated granulocytes and of whole blood from dogs was evaluated. Chemiluminescence of whole blood samples created an undesired quenching effect by the red blood cells which makes the assay difficult to apply in pathological cases with low formation of oxygen metabolites. This problem was avoided when chemiluminescence was determined, using isolated granulocytes. A cell concentration of 5 x 10(9)/l was needed to create optimal conditions. The Boyden chamber technique was used for study of random migration and chemotaxis. Casein (0.1%), zymosan activated serum with and without epsilon-amino-n-caproic-acid and homologous serum were effective chemoattractants for canine granulocytes, while FMLP (formyl-methionyl-leucyl-phenylalanin) did not attract canine granulocytes.

Phagocytosis induced by yeast cells in canine granulocytes: a methodological study.

A phagocytic function assay of canine granulocytes was established. This method allows the proportion of active granulocytes to be estimated as well as the number of adhered and ingested yeast cells. The influence of different factors on phagocytosis was studied. Temperature variation within the interval 36-41 degrees C did not affect phagocytosis. The incubation time for optimal phagocytosis of yeast cells was 35 min. The opsonization procedure giving the optimal phagocytosis was purified IgG and serum together.

Cardiac manifestation and blood catecholamine levels during succinylcholine-induced stress of malignant hyperthermia sensitive pigs.

Fifteen crossbred pigs of Swedish Landrace and Yorkshire, about 6 months of age and susceptible to develop malignant hyperthermia (MH) when exposed to halothane, were subjected to stress provoked by the myorelaxant succinylcholine. The results were compared with those of 12 normal pigs. During the stress the halothane-sensitive (HS) pigs showed much higher levels of plasma noradrenaline and adrenaline and more severe ventricular arrhythmias than the controls. The degree of myocardial degeneration and necrosis being similar to catecholamine induced myocardial damage was significantly higher in the HS pigs than in the controls. The ultrastructural examination revealed three main types of changes in affected myocardial cells. One type of myocardial cell damage was characterized by various degree of hypercontraction, enlarged mitochondria with dense bodies and dilated sarcoplasmic reticulum. The other type showed mitochondria with tubular configuration whereas the third type of cell damage was characterized by almost normal mitochondria combined with a severe damage of the myofilaments. Three HS pigs which died within 30 min after stress showed signs of malignant hyperthermia. No signs of the disease were observed in the other 12 HS pigs.

Catecholamine-induced free radicals in myocardial cell necrosis on experimental stress in pigs.

Normal pigs, crossbreeds of Swedish Landrace and Yorkshire, about 6 months old, were subjected to experimental stress, induced by the myorelaxant succinylcholine, for 12 min. Besides one group of control pigs, one group of pigs were pretreated with alpha-tocopherol (vitamin E) combined with selenium (Tokosel Vet) injected i.m. every second day for 11 days, and another group was given zinc (ZnSO4.7H2O) in the fodder for 1 month plus one injection i.p. 2 days before the stress. The stress-induced heart lesions, morphologically graded according to evaluation scores, were significantly reduced in both the pretreated groups when compared with the control pigs. The blood levels of catecholamines (CA) were increased to about the same degree in the three groups during the stress. The protection observed is suggested to be due to the fact that vitamin E, selenium and zinc are involved in systems acting as scavengers of free radicals. The present results together with earlier ones are discussed to support the CA-hypothesis for stress-induced heart lesions: some types of stress can increase the sympathetic activity to such an extent that released CA, via beta-adrenoceptor mechanisms affects the cell metabolism to such a degree that cytotoxic free radicals are formed, producing myocardial cell necrosis.

Sepsis in sheep reduces pulmonary microvascular sieving capacity.

The changes in pulmonary microvascular permeability in sheep, after infusion of live Escherichia coli, were studied using estimations of the osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M and based on these estimations equivalent pore dimensions were calculated. A chronic lung lymph fistula was prepared in seven sheep. After a base-line period, left atrial pressure (Pla) was increased. E. coli (10(9) X kg body wt) were given after attaining filtration independent L/P values. The sigma's for the normal lung were calculated to 0.73 for total protein and to 0.65, 0.76, and 0.91 for albumin, IgG, and IgM, respectively. The equivalent pore radii were determined to 50 and 175 A with 35% of the filtration accounted for by the large pores. After bacterial infusion, the sigma's for total protein, albumin, IgG, and IgM decreased significantly from preseptic values to 0.58, 0.50, 0.64, and 0.83, respectively. After sepsis the small pores were 50 A and the large pores 200 A with 49% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large-pore number increased 32% after bacterial infusion. These results indicate that pulmonary microvascular permeability may have increased due to the sepsis.

The pulmonary microvascular response to infusion of live Escherichia coli in sheep with acutely or chronically prepared lung lymph fistula.

The effects of infusion of live Escherichia coli bacteria in awake sheep with a chronic lung lymph fistula (n = 15) were compared to anesthetized animals (n = 7) receiving the same septic insult after surgical trauma including bilateral thoracotomies for lung lymph cannulation (acute group). During preseptic baseline conditions, pulmonary arterial pressure (Ppa) and central venous pressure (Pcv) were increased and leukocytes decreased in the newly operated animals compared to the sheep with a chronic lung lymph fistula. After i.v. infusion of live E. coli 10(9) X kg-1 b.w. over 20 min, arterial pressure (Psa), cardiac output (Qt), leukocytes, and partial pressure of arterial oxygen (PaO2) decreased in both groups. Ppa peaked after 15 min at 37.2 +/- 2.5 in the chronic and 33.4 +/- 3.3 mm Hg in the acute group. In the chronic group, Ppa remained elevated but not in the acute group during the rest of experiment. Lung lymph flow (QL) increased significantly in both groups during the initial high Ppa, but it increased to a higher level in the chronic group. After 150 min, QL did not differ between the groups but remained elevated over baseline. Lymph-to-plasma concentration ratio (L/P) for total protein decreased in the chronic group during the initial high QL. This decrease was not seen in the acute group that had a significantly higher L/P between 30 and 120 min after sepsis. The high QL with unchanged L/P compared to baseline indicated increased permeability in the pulmonary microvessels in both groups but the changes in permeability, hemodynamics, or respiratory parameters after sepsis were not aggravated by the surgical trauma.

Effect of phenoxybenzamine on myocardial cell necroses and blood levels of catecholamines in pigs subjected to stress.

The degree of myocardial cell necroses after stress was investigated in 6 pigs, weighing 70 to 90 kg. The stress was induced by a myorelaxant, succinylcholine, for about 12 min. The alpha-adrenoceptor blocking agent phenoxybenzamine (PBZ) was given orally 3 times a day in a total dose of 50 to 60 mg per day for 6 to 7 days before the stress. The plasma levels of noradrenaline and adrenaline were assayed in blood samples drawn before, during, and immediately after the period of stress. Heart cell necroses were found in all the cases. The activity of the sympathetic-adrenomedullary system, indicated by the plasma levels of catecholamines, was intense during the stress. When the present results were compared with those of earlier studies the degree of heart cell necroses was significantly smaller after PBZ in control pigs but significantly higher than after the beta-adrenoceptor blocking agent propranolol given for about a week. PBZ exerts a number of different actions, as briefly discussed, some of them appearing to be harmful for the heart during stress while others appear to be protective for the heart.

DNA binding proteins in canine sera. A method for removal of nonspecific DNA binding in the Farr assay.

A panel of canine sera, the majority of which were collected from clinically healthy dogs, were investigated for antibodies against double stranded (dsDNA) by the Farr radioimmunoassay technique. Non-specific DNA binding agents interfering with the Farr assay were detected in all sera. Heat inactivation at 60 degrees C or treatment with dextran sulphate was shown to eliminate this kind of unspecific DNA binding while not affecting true antibodies to dsDNA. Canine sera positive in the Farr assay after inactivation at 60 degrees C were positive also in immunofluorescence for anti-nuclear antibody on rat liver sections and for dsDNA with Chrithidia luciliae as antigen preparation. IgG or glycoprotein nature of the non-specific DNA binding could be excluded by means of affinity chromatography on protein A and the lectin lentil.

Studies of serum-transferrin and some hematologic parameters in vitamin E and selenium deficient pigs.

Sows of Swedish Landrace x Yorkshire breed were fed a diet extremely deficient in vitamin E, selenium (VESD) during the last six weeks of pregnancy and compared to sows of the same breed and age fed a normal commercial diet. The transferrin concentration in serum and different hematological parameters were estimated at regular intervals in the sows before and after partus and in their piglets at birth and during the first four weeks of life. The transferrin concentration in the serum of piglets from sows fed the VESD diet did not differ from the corresponding values in normal pigs whereas the VESD piglets showed significantly lower hemoglobin values at birth and higher MCV values during the first three weeks of life than the normal piglets. A disturbance of the erythropoesis in these piglets due to vitamin E/selenium deficiency is discussed.

Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera.

The use of protein A from S. aureus (SpA) as an anti-IgG reagent in immunological techniques has extended in recent years, together with knowledge about its interaction with immunoglobulins of different species. Current data with respect to the binding of protein A to immunoglobulins and to the levels of immunoglobulins in the sera of some mammalian species are reviewed.

Creatine kinase isoenzymes in serum of pigs having myocardial and skeletal muscle necrosis.

Serum creatine kinase (CK) and lactic dehydrogenase (LD) isoenzyme activities were measured in blood serum of pigs having myocardial damage and skeletal muscular lesions. Myocardial and muscular damage was induced by restraint stress provoked by intravenous infusion of a pharmacological restraint (succinylcholine-chloride) during 12 minutes. Pigs of Swedish Landrace and Swedish Landrace X Yorkshire breed, stress-susceptible (halothane-sensitive) and nonreacting pigs were studied. Severe myocardial damage and slight to moderate skeletal muscle necrosis were found 24 hours after restraint stress in the stress-susceptible pigs whereas in nonreacting pigs generally only myocardial lesions of moderate extent were registered. No significant increase was detected in the serum CK-BB (CK-1) or CK-MB (CK-2) activity whereas a pronounced elevation of the CK-MM (CK-3) activity was found, particularly in the stress-sensitive animals. In the myocardial tissue of pigs only a low CK-MD activity was found (about 4-5% CK-MD in addition to CK-MM) and this may explain the low CK-MB activity in serum of pigs subjected to severe myocardial damage. This is further supported by the pronounced increase in the anodal serum fractions LD 1-2 in animals free from skeletal muscular lesions. In the halothane-sensitive pigs skeletal muscle necrosis besides the myocardial lesions contributed to the high levels of CK-MM activity in serum.