An experimental soft-release of oil-spill rehabilitated American coots (Fulica americana): II. Effects on health and blood parameters.

The Unocal-Metrolink oil spill of 21 February 1995 resulted in approximately 7800 barrels of San Joaquin crude oil being deposited into the San Gabriel River in Huntington Beach, CA, USA. In order to determine long-term pathological effects of oil exposure and rehabilitation, hematological and serum biochemical parameters for both rehabilitated (RHB) American coots (Fulica americana) and reference (REF) coots were examined every 3-4 weeks (56, 81, 108 and 140 days post oil exposure) after birds were cleaned, rehabilitated and soft-released. Most significant differences in monthly comparisons between RHB and REF birds occurred 56 days following oil exposure. Total white blood cell (WBC) count, albumin:globulin (A:G) ratio and calcium concentration were higher in RHB birds compared to REF birds 56 days post oil exposure. In addition, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and creatine kinase activities, and creatinine, total protein (TP) and globulin concentrations were lower in RHB birds. Blood results from 56 days post oil exposure for RHB coots which subsequently died were compared to blood results from days 108 and 140 for REF coots which survived. Oiled and rehabilitated birds which died had significantly higher WBCs, packed cell volume, TP and globulin concentrations, and lower A:G ratio, MCH, MCHC, glucose and sodium concentrations compared to REF birds which survived. Blood result differences detected at 3-4-week intervals between RHB and REF survivors, and differences detected between RHB coots which died and REF coots which survived, suggested that RHB coots developed an inflammatory response (infectious or non-septic) and, concurrently, may have experienced decreased immune responsiveness. Additionally, RHB coots experienced either an iron (Fe) utilization or Fe metabolism problem. These pathophysiological mechanisms were consistent with increased hemosiderin (stored Fe) present in the liver, spleen and kidney of necropsied RHB birds, and may have contributed to RHB coot mortality. When blood parameter differences were examined for their impact on survival time, it was determined that RHB coots had shorter survival times if they had very high cholesterol (> or =449 mg/dl) or chloride (> or =110 MEQ/l) concentrations on day 56 post oil exposure. Interestingly, the lack of differences between RHB and REF coots from day 81 through day 140 suggested that, from a hematologic and clinical chemistry perspective, coots which were oiled, rehabilitated, released and survived at least 3.5 months could not be differentiated from wild (REF) coots. From these findings it appears that blood analysis, coupled with post-release survival data, may help discern reasons for increased mortality of oiled and rehabilitated birds, compared to non-oiled reference birds.

Hematology and clinical chemistry of cyprinid fish. Common carp and goldfish.

Evaluation of the clinical status in aquatic species is compromised by the limited diagnostic techniques that can be performed in these species. The hematologic and plasma chemistry parameters can provide predictive information, although these parameters can be highly variable owing to the influence of various intrinsic and extrinsic factors. However, these parameters are fairly stable in acclimated, well-managed fish if stress is reduced during collection and samples are properly collected and analyzed. Evaluation of any single parameter is not predictive and, therefore, not recommended. Ideally, the diagnostic protocol should include evaluation of the hematologic indices, total and differential cell counts, TPP, glucose, sodium, and chloride. The practitioner who is routinely involved in aquatic animal medicine should consider in-house evaluation of these parameters.

Physiological responses of fathead minnow larvae to rice pesticides.

Newly hatched fathead minnow (Pimephales promelas) larvae were exposed for 4 days to two pesticides and ambient receiving waters to simulate conditions in the Sacramento River, California, during the striped bass spawning season which coincides with pesticide use in adjacent rice culture. Carbofuran and molinate were tested at two concentrations: a higher level approximating one-half the LC50 and a level much lower that is similar to that seen in the receiving waters of Colusa Basin Drain. Physiological measurements were made immediately after the exposures and again after a 10-day recovery period in noncontaminated waters. These included growth rate, swimming capacity, response to a mild electric shock, upper and lower lethal temperatures, and activity of acetylcholinesterase in whole-body homogenates. The higher concentrations of carbofuran and molinate caused reductions in swimming capacity, an increased sensitivity to the electric shock, and a reduction in upper lethal temperature. Acetylcholinesterase was reduced in those larvae exposed to the higher levels of carbofuran. In general, the lower levels of pesticide exposure caused no measureable effects nor did exposure to water from Colusa Basin Drain.

Dysfibrinogenemia or afibrinogenemia in a Border Leicester lamb.

Hereditary fibrinogen deficiency is a rare condition in all species. Measurement of plasma fibrinogen should indicate low levels. Specific factor assays and pedigree analysis are essential in establishing a definitive diagnosis of the hereditary deficiency. Differentiation between afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia requires sophisticated techniques and assistance from a specialized laboratory.

Cytokeratin-filament expression in epithelial and non-epithelial tissues of the common carp (Cyprinus carpio).

Cytokeratin expression in mammals is generally restricted to epithelial cells and has been utilized to differentiate epithelial from nonepithelial tissues in these species. Since cytokeratins have been shown to be highly conserved during vertebrate evolution, the objective of the present study has been to ascertain the expression pattern of cytokeratins in tissues of the common carp (Cyprinus carpio). A panel of 10 anti-human cytokeratin antibodies was evaluated using a streptavidin-biotin-peroxidase complex detection system. Tissues were fixed in 10% neutral-buffered formalin, 100% ethanol or methacarn. Only formalin-fixed tissues were pre-digested with trypsin prior to immunostaining. Formalin-fixed tissues generally resulted in a less intense, more diffuse staining pattern with considerable background compared with ethanol and methacarn and was therefore the least desirable fixative. The diverse staining pattern observed with the various antibodies used in this study was consistent with previous findings in other teleosts. The results confirm that cytokeratin expression in teleosts is fundamentally different from that in mammals and therefore should be used as a method to differentiate epithelial cell types in these species only with discretion.

Haematological, bone marrow and clinical chemical changes in neonatal foals given canine recombinant granulocyte-colony stimulating factor.

Five Thoroughbred and Quarter Horse cross foals were given 20 micrograms canine recombinant granulocyte-colony stimulating factor (rcG-CSF) per kg bwt intramuscularly (i.m.) on the day of birth and 10 micrograms rcG-CSF/kg for 13 additional days. During this time and for an additional 21 days haematology, bone marrow and clinical chemical analyses were performed. After one day of rcG-CSF administration leucocyte and neutrophil counts increased from 9.16 x 10(9)/l to 23.44 x 10(9)/l and from 6.45 x 10(9)/l to 19.61 x 10(9)/l, respectively. The counts continued to increase for the next 3-4 days and then there was a slight decrease. A second increase followed and the leucocyte and neutrophil counts increased to 52.84 x 10(9)/l and 45.16 x 10(9)/l on the day after the last rcG-CSF administration (Day 15). The counts decreased rapidly immediately after the administration of rcG-CSF was stopped and then at a slower rate. The cell counts were still higher than in the controls at the end of the study period (Day 35). Bone marrow cellularity increased from 10-25% before rcG-CSF was given to 60-80% after 5 days. The increase in cellularity was due to increased myeloid activity because the myeloid to erythroid ratio increased from 2.7 to 8.8. Serum chemistry changes were minimal although foals given rcG-CSF at various times had lower glucose concentrations and increased alkaline phosphatase and gamma glutamyl transferase activities.

Modifications of a cholinesterase method for determination of erythrocyte cholinesterase activity in wild mammals.

A method to determine erythrocyte cholinesterase (ChE) activity was modified for use in wild mammals. Erythrocyte ChE of California voles (Microtus californicus) was primarily acetylcholinesterase (AChE), which was similar to the brain and unlike plasma which was primarily butyrylcholinesterase (BChE). Triplicate erythrocyte AChE analyses from individual animals of several species of wild rodents revealed a mean coefficient of variation of 8.7% (SD = 4.3%). Erythrocyte ChE activity of several wild mammals of California revealed that mule deer (Odocoileus hemionus) had the highest erythrocyte AChE activity (1,514.5 mU/ml) and dusky-footed woodrats (Neotoma fuscipes) had the lowest activity (524.3 mU/ml). No ChE activity was found in erythrocytes of several species of birds and fish.

Sublethal effects of three pesticides on Japanese medaka.

One- to 2-day-old medaka (Oryzias latipes) larvae were exposed for 4 days to the rice field pesticides methyl parathion, molinate, carbofuran and a mixture of all three. Pesticide concentrations were one-half the 96 h LC50 ("high concentration") and levels approximating those measured in receiving waters from rice field runoff ("low concentration"). Maximum swimming speed, spontaneous muscular activity, acetylcholinesterase activity, dry weight, RNA:DNA ratio, and five morphometric variables were determined at the end of the exposures. Larvae were retained for an additional 10 days in non-contaminated water, and the same measurements taken to investigate residual effects. Results are compared to a parallel study on striped bass larvae to evaluate the suitability of this species as a surrogate for the bass in toxicological studies involving sublethal exposures. There was no relationship between mortality rate and pesticide exposure either during the exposures or during the ten day subsequent period. Only the high concentration of carbofuran caused an impairment of swimming performance. Spontaneous activity was stimulated in the high concentration of molinate and the combined pesticides groups. Acetylcholinesterase was severely inhibited in parathion and molinate, and this persisted in some cases after 10 days in non-contaminated water. The pesticides had little effect on growth rate except for molinate which acted as a stimulant. Combining the three pesticides caused a less than additive effect. Except for decreases in acetylcholinesterase, the sublethal effects of the pesticides tested at the very low concentrations used were subtle. Apparently, larvae of this species are less sensitive to these pesticides than are striped bass larvae.

Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.

Evaluation of the haemostatic profile in the pre- and post parturient mare, with particular focus on the perinatal period.

Various haemostatic analytes were systematically evaluated for four months pre-partum and five months post partum in 14 healthy mares. The plasma fibrinogen concentration and both Factor VIII:C and von Willebrand factor activity showed gradual increases from mid-gestation and reached maximal, or near maximal activity at parturition. These increases were paralleled by an increase in plasma fibronectin concentration, the appearance of fibrinogen degradation products, and a modest rise in antithrombin III concentration. In contrast, the activity of Factor VII and Factor IX, and the one-stage prothrombin (PT) time and the activated partial thromboplastin (APTT) time remained relatively constant throughout the pre- and post parturient period.

Heterogeneity in phagocytic and nitroblue tetrazolium reductive properties of neutrophils from cows.

Phagocytic and nitroblue tetrazolium (NBT) reductive activities of blood neutrophils from 19 Holstein heifers were measured by light microscopic and spectrophotometric methods, respectively. These functional properties of neutrophils correlated well (r = 0.64) and varied significantly (P less than 0.05) among animals studied. Variations in phagocytosis and NBT reductive activities attributable to the source of sera were determined in experiments in which cells from the same cows and zymogen particles opsonized with heat-inactivated autologous or homologous sera were used. Variations attributable to the source of cells were determined in experiments in which cells from different cows and particles opsonized with pooled sera from all the cows were used. Most of the variation in phagocytic properties and NBT reductive activities was attributable to the source of cells (ie, each cow). The source of sera contributed slightly to the variation in NBT reductive activities, but not to the phagocytic properties. These results support the concept of functional heterogeneity of neutrophils among cows.

Reference ranges and the influence of age and sex on hematologic and serum biochemical values in donkeys (Equus asinus).

Hematologic and serum biochemical values were determined in blood samples from 217 donkeys (Equus asinus). Donkeys were classified on the basis of size, sex, age, and whether they were domestic or feral. Parametric (mean +/- 2 SD) and nonparametric (2.5th to 97.5th percentile) reference ranges were calculated for each analyte. For all donkeys, 26 of 46 analytes significantly departed from gaussian distribution. Serum lactate dehydrogenase activity in miniature donkeys was higher than that in other donkeys. Differential leukocyte counts in feral donkeys differed from those in other types in ways that suggested that the former had smaller parasite loads or experienced greater stress. Erythrocyte, lymphocyte, and platelet counts and fibrinogen, glucose, inorganic phosphorus, and potassium concentrations decreased with age. Eosinophil counts, mean corpuscular volume, and plasma protein, serum protein, and serum globulin concentrations increased with age. Female donkeys had significantly (P less than 0.05) higher mean corpuscular hemoglobin concentration and leukocyte and neutrophil counts than did male donkeys. Mean corpuscular hemoglobin increased with age, and females had higher values than did males of all age groups. An interaction between age and sex was observed for alkaline phosphatase activity, with a trend for decreased activity with age.

Reference ranges for hematologic and serum biochemical values in llamas (Lama glama).

Hematologic and serum biochemical values were determined in 174 llamas of all age groups and both sexes from ranches in California and Nevada. Compared with hematologic values for horses and cattle, llama erythrocytes were more numerous (10.1 to 17.3 x 10(6)/microliters), but the PCV was lower (25 to 45%) because the smaller elliptical cells pack tighter. The mean corpuscular volume was half that of horses and cattle (22 to 29.5 fl). The mean corpuscular hemoglobin concentration was higher (38.9 to 46.2 g/dl), and the mean corpuscular hemoglobin slightly lower (9.6 to 12.6 pg). Most serum biochemical values were similar to those of cattle and horses, with the exception of triiodothyronine (48 to 468 ng/dl) and thyroxin (9.8 to 30 micrograms/dl), which are up to 10 times higher than values for other domestic species.

Ultrastructural analysis of platelets and megakaryocytes from a dog with probable essential thrombocythemia.

Blood platelets and bone marrow megakaryocytes from a dog with essential thrombocythemia were analyzed by transmission electron microscopy and compared with those of control dogs. Platelets varied in size and shape and contained enlarged and dilated open canalicular systems. Megakaryocytes were dysmorphic and had evidence of abnormal maturation, with large numbers of megakaryoblasts containing alpha granules and poorly organized and reduplicated demarcation membranes. The fewer, more mature megakaryocytes lacked normal cytoplasmic organization. Most notable was the absence of well-demarcated "platelet fields," due to the excessive and disorderly array of demarcation membranes.

Probable essential thrombocythemia in a dog.

Essential thrombocythemia (ET) in an 11-year-old dog was characterized by persistently high platelet counts (range, 4.19 X 10(6)/microliters to 4.95 X 10(6)/microliters, abnormal platelet morphology, marked megakaryocytic hyperplasia in the bone marrow, absence of circulating megakaryoblasts, and history of splenomegaly and gastrointestinal bleeding. Increased numbers of megakaryocytes and megakaryoblasts (15% to 20%) in the bone marrow were confirmed by a positive acetylcholinesterase reaction. Another significant finding was the presence of a basophilia in blood (4,836/microliters) and bone marrow. The marked persistent thrombocytosis, absence of reactive (secondary) thrombocytosis, abnormal platelet morphology, and quantitative and qualitative changes in the megakaryocytic series in the bone marrow suggested the presence of a myeloproliferative disease. Cytochemical and ultrastructural findings aided in the diagnosis of ET. The dog was treated with radiophosphorus. The results was a rapid decline in the numbers of megakaryoblasts and megakaryocytes in the bone marrow and platelets and basophils in the peripheral blood. The dog died unexpectedly of acute necrotizing pancreatitis and diabetes mellitus before a complete remission was achieved.

Phagocytic and postphagocytic activities of bovine neutrophils for pure and mixed bacterial cultures.

A comparative study of phagocytosis and postphagocytic oxidative metabolic activity of bovine blood neutrophils incubated with pure and mixed cultures of Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae was preformed. Most neutrophils when incubated with mixed cultures showed preferential phagocytosis for one species and a smaller number phagocytized both species of microorganisms. Percent phagocytosis for E. coli in pure culture was similar to that of Strep. agalactiae in pure culture and higher than that for Staph. aureus in pure culture. Neutrophils incubated with mixed cultures of E. coli and Staph. aureus or E. coli and Strep. agalactiae showed greater than expected phagocytosis of each microorganisms alone and reduced phagocytosis of both microorganisms together. Postphagocytic oxidative metabolic activity of neutrophils, measured by percent nitroblue tetrazolium reduction, did not differ following phagocytosis of these three microorganisms in pure cultures. In comparison, a synergistic effect on nitroblue tetrazolium reductive activity was seen in mixed cultures as evidenced by higher percent nitroblue tetrazolium reduction following phagocytosis of both E. coli and Staph. aureus or E. coli and Strep. agalactiae. These observations indicate that the phagocytic and metabolic activities of neutrophils for bacteria in mixed cultures may not be identical to those in pure cultures.