Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs.

To clarify the immunologic effects of vaccination in dogs, we monitored total leukocyte and lymphocyte counts, humoral antibody responses, blastogenic responses of lymphocyte, and delayed type hypersensitivity (DTH) responses after vaccination. Mixed vaccines were administered on day 0 except for canine parvovirus (CPV) vaccine which was readministered on day 21. The puppy and adult dogs had a significant decrease in leukocyte and lymphocyte counts on day 7. The puppies showed a significant increase in the blastogenesis of lymphocytes after each vaccination, whereas the adult dogs had no significant changes. However, the adult dogs were divided into two groups, high responders and low responders in blastogenesis of lymphocytes. The dogs with higher or lower response in SI values on day 0 tended to show decrease or increase after the first vaccination, respectively. Since almost all dogs developed high titers of humoral antibody, it is considered that vaccination acts in an immunomodulative fashion. DTH responses to phytohemagglutinin (PHA) and CPV vaccine monitored at 0, 3, and 8 weeks after the first vaccination produced strong reactions, in particular those to CPV vaccine rose significantly after vaccination and maintained the higher responses for at least 2 months. These results suggest that DTH responses to PHA and CPV vaccine are helpful to monitoring non-specific and specific immune functions in vivo, therefore, DTH could be used as simple and rapid immunologic tests in canine practice.     

Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy

Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.     

Graft-versus-host disease in severe combined immunodeficiency/beige mice administered canine leukocytes

This report describes the development and lesions of graft-versus-host disease (GVHD) in severe combined immunodeficiency/ beige (SCID/BG) mice after the administration of canine leukocytes. Intraperitoneal injections of 0.87 x 10(7) canine lymphocytes were given to each of 9 mice; 5 mice received no canine lymphocytes. Morphologic evidence of successful engraftment included peritoneal aggregates of lymphocytes and repopulation of spleen and lymph nodes by lymphocytes. Canine CD45R was expressed by 2.25% of peripheral blood leukocytes in the 1 mouse tested 65 d after engraftment but by none of the cells of a control mouse. Canine immunoglobulin G was detected in serum samples from 5 of the 6 tested mice given canine lymphocytes but none of the control mice. By 13 to 65 d after receiving canine lymphocytes, 5 of the 9 mice had died of GVHD or had been euthanized because of it; all the control mice remained healthy. Lesions of GVHD included hemolytic anemia, cholangiohepatitis, alveolitis, and disseminated intravascular coagulation. Serum from the donor dog and from all 15 randomly selected dogs caused agglutination of normal mouse erythrocytes, supporting a diagnosis of immune-mediated hemolytic anemia in the dog-mouse chimeras. All of the mouse serum tested contained murine immunoglobulin, and this "leakiness" may have contributed to the development of GVHD.     

Hematologic and serum biochemical effects of long-term administration of anti-inflammatory doses of prednisone in dogs.

Results of routine hematologic and serum biochemical analyses from 12 healthy adult male dogs that were given prednisone (0.55 mg/kg of body weight, PO, q 12 h) for 35 days were compared with those of a control group of 6 dogs that were given gelatin capsules. Analyses were performed at 2-week intervals during and after prednisone administration. Lymphocyte and eosinophil counts were significantly (P less than 0.005) decreased after 2 and 4 weeks of prednisone treatment, compared with controls. Two weeks after treatment, eosinophil counts in prednisone-treated dogs were similar to those of control dogs, whereas lymphocyte counts remained low 4 weeks after treatment in treated dogs (1,869 +/- 145 cells/microliters), compared with that in control dogs (3,662 +/- 548 cells/microliters). Neutrophil and monocyte counts did not significantly change during glucocorticoid administration. Mean platelet volume significantly (P less than 0.001) decreased after 4 weeks of prednisone treatment, but returned to pretreatment values by 2 weeks after treatment. Four weeks of prednisone treatment did not cause significant increased activity in serum alanine transaminase, total alkaline phosphatase or the steroid-induced isoenzyme of alkaline phosphatase. Significant increases in serum albumin (P less than 0.001) and total protein (P less than 0.05) concentrations were detected after 4 weeks of treatment, but mean values were not significantly different from those of controls 2 weeks after treatment ended. Results of our study indicate that eosinophil and lymphocyte counts are the most sensitive indicators of long-term glucocorticoid administration at anti-inflammatory dosages of 1.1 mg/kg daily.     

Survey of arabian foals in poland for immune system disorders

During the foaling seasons of 1983 and 1984, 228 (76%) of 300 Arabian foalsborn in Poland were analyzed for immune system disorders by performing leukocyte differential counts and by quantitating serum concentrations of IgM and IgG. IgM concentrations and absolute lymphocyte counts were within normal limits for all foals tested. Twelve foals (5.3%) demonstrated failure or partial failure of colostral IgG transfer (foal serum IgG < 400 mg/dl). All 12 foals survived. No cases of combined immunodeficiency, selective IgM deficiency or agammaglobulinemia were detected among the 228 foals tested.     

Survey of serum IgA, IgG, and IgM concentrations in a large beagle population in which IgA deficiency had been identified

Concentrations of serum IgA, IgG, and IgM were determined for 829 adult Beagles from a commercial kennel in which several IgA-deficient dogs had been identified previously (index kennel). These values were compared with measurements of 100 adult dogs from another Beagle kennel (control kennel). After adjustment for differences in the ages and gender of the dogs, dogs from the index kennel had significantly (P less than 0.0001) lower IgA concentrations (mean, 46 mg/dl) than dogs from the control kennel (mean, 68 mg/dl). Regardless of kennel, males had significantly (P less than 0.01) higher IgA concentrations than did females. Dogs in the control kennel had significantly (P less than 0.04) higher IgG concentrations (mean, 2,649 mg/dl) than did dogs in the index kennel (mean, 2,478 mg/dl), and female dogs in the control kennel had significantly (P less than 0.05) higher IgM concentrations (mean, 189 mg/dl) than dogs of either sex in the index kennel (mean, 162 mg/dl) or male dogs in the control kennel (mean, 163 mg/dl). For both sexes, concentrations of IgA, IgG, and IgM increased with age.     

Potential effects of glucocorticoids on serum iron concentration in dogs

Prednisone was give norally(2mg/kg b.i.d.) to seven healthy mixed breed dogs for 3 consecutive days. Serum iron concentration increased significantly (p < 0.05) from 142 +/- 26 micro g/dl (mean +/- SE) before a drug adminis- tration on Day 0 to a maximum of 307 +/- 47 micro g/dl on Day 2, and returned to the Day 0 value by Day 5. Mean total iron binding capacity did not vary more than 25% from the Day 0 value during the 9 day long study. The percent saturation of transferrin with iron increased from 33 +/- 6% on Day 0 to a maximum of 71 +/- 9% on Day 3. This determination had decreased to 34 +/- 3% on Day 5. No statistically significant changes occurred in these parameters studied in six control dogs that were not given the drug. To determine whether serum iron concentration might be correlated with endogenous serum cortisol concentration, these tests were determined in serum collected from nine dogs at 7 a.m., 3 p.m., and 11 p.m. each day for 3 consecutive days. Serum iron concentration was lower at 7 a.m. (147 +/- 9 micro g/dl) than at 3 p.m. (164 +/- 9 micro g/dl) or 11 p.m. (159 +/- 10 micro g/dl). Likewise serum cortisol was lower at 7 a.m. (1.29 +/- 0.18 micro g/dl) than at 3 p.m. (1.49 +/- 0.19 micro g/dl) or 11 p.m. (1.51 +/- 0.22 micro g/dl). There was a significant positive linear correlation between serum iron and serum cortisol concentrations when they were compared using mean values for each dog. From these studies, it appears that exogenously administered glucocorticoids and endogenous increases in serum cortisol concentrations may result in increased serum iron concentrations in dogs.     

Transient leakage across the blood-cerebrospinal fluid barrier after intrathecal metrizamide administration to dogs

Cerebrospinal fluid samples from 9 dogs given 84 mg of metrizamide/kg of body weight intrathecally were collected at intervals from 3 hours to 30 days after treatment and were compared with CSF samples collected before metrizamide treatment (base line) and with samples taken at similar intervals from 2 nontreated control dogs. Increased CSF albumin (mean 19.2 mg/dl) and immunoglobulin (Ig) G (mean 5.91 mg/dl) concentrations occurred 1 day after myelography, compared with base-line concentrations (6.15 and 1.24 mg/dl, respectively) and with concentrations from controls. Immunoglobulin M and IgA concentrations also were increased in some of these samples. However, immediately after myelography (3 hours after treatment) CSF albumin and IgG concentrations were comparable with those of controls, and these values returned to base line within 5 days of myelography and remained so for 30 days. A high correlation between albumin and IgG concentrations of individual CSF samples indicated the likelihood that leakage across the blood-CSF barrier was the origin of the increased values. A transient increase in CSF leukocytes, consisting of mononuclear cells and neutrophils, was also noticed 1 day after treatment but not at other times and not in controls. Nonsuppurative, predominately histiocytic meningitis of decreasing intensity was noticed in dogs euthanatized at 1 or 5 days, but not in dogs euthanatized at 10, 20, or 30 days after treatment. The meningeal cells stained intensely with periodic acid-Schiff stain and intracellular contrast medium was ultrastructurally apparent in phagolysosomes. Control dogs killed after 30 days did not have these changes. The intrathecal administration of metrizamide resulted in transient leakage of serum components into CSF and an accompanying transient meningitis.     

Age-associated changes in the immune system of German shepherd dogs

In order to look into the ageing of the canine immune system we investigated age-related changes and associated gender-related differences in parameters of innate and acquired immunity in German Shepherd dogs. We obtained the following findings: white blood cell counts, peripheral blood lymphocytes, lymphocyte proliferative activity and interleukin-2 (IL-2) serum concentrations were significantly lower in the group of old animals, whereas the concentrations of gamma-globulins and the functional activity of the complement system were significantly higher in the elderly. Phagocytic and bactericidal activity of polymorphonuclear cells, as well as their 'killing function,' the serum cytokine-like activities of tumour necrosis factor-alpha and the plasma concentrations of immunoglobulin G, as well as of alpha- and beta-globulins, were not significantly affected by age, whereas natural killer-cell activity and the serum cytokine-like activities of IL-1 were significantly higher only in the group of female old animals. With regard to gender-related differences, lymphocyte proliferative activities as well as plasma concentrations of alpha-globulin were significantly higher in the group of female animals, whereas the absolute numbers of segmented neutrophils were significantly lower. Species analogies with regard to ageing as presumed to exist between man and laboratory rodents also seem to be applicable to the dog. The observed age-related changes in the canine immune system are probably among the main causes for the multimorbidity of old age, affecting life expectancy and mortality in the dog and should be recognized and considered by the attending veterinarian.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Abnormalities of Serum Magnesium in Critically III Dogs: Incidence and Implications

Serum magnesium (Mg) is an infrequnetly measured electrolyte in small animal patients. Currently, little is known about the prevalence and significance of abnormalities in serum Mg in animals. Therefore, a prospective study was performed to examine the incidence and clinical implications of abnormalities in serum Mg levels in critically ill dogs.
Serum Mg and other electrolytes were measured in 93 normal dogs housed at the Purina Pet Care Center and in 48 ill dogs admitted to a small animal critical care unit. The normal reference range for canine serum Mg was determined to be 1.89 – 2.51 mg/dl. Based on this range, 54% of the critically ill dogs were hypomagnesemic (< 1.89 mg/dl) and 13% were hypermagnesemic (> 2.51 mg/dl). Of the electrolytes measured in these patients, serum Mg had the highest prevalence of abnormal values. Hypomagnesemic patients had a significantly higher incidence of concurrent hypokalemia and hyponatremia (p < 0.05), as well as a longer length of hospitalization (p < 0.05) than their normomagnesemic counterparts. Hypermagnesemic patients were 2.6 times more likely not to survive their illness when compared to patients with normal serum Mg levels.
Abnormalities in serum Mg appear to be common in critically ill dogs. These patients commonly have other concurrent electrolyte abnormalities. Since serum Mg is not routinely measured, the presence of hypokalemia or hyponatremia should alert the clinician to the possibility of coexisting hypomagnesemia. The clinical implications of hypomagnesemia and hypermagnesemia in ill dogs appear to involve prolonged hospitalization and increased mortality, respectively: however, the exact etiology remains undetermined.     

Lymphocyte transformation suppression caused by pyoderma--failure to demonstrate it in uncomplicated demodectic mange

Three dogs with demodectic mange uncomplicated by a bacterial infection and 9 dogs with demodectic mange and pyoderma were tested for their lymphocyte response to phytomitogens in vitro and for the presence of the serum's lymphocyte immunoregulatory factors (SLIF) suppressing blastogenesis. None of the 3 dogs with uncomplicated demodectic mange showed any detectable dysfunction of their lymphocytes or presence of the blastogenesis suppressing SLIF. Their lymphocytes generally responded to the mitogens with more blastogenesis than lymphocytes from healthy controls. On the other hand, in the group of 9 dogs with demodicosis complicated by a bacterial infection, high levels of the blastogenesis suppressing SLIF for concanavalin A-sensitive cells were detected in 4 dogs, for phytohemagglutinin-sensitive cells in 2 dogs, and for pokeweed mitogen-sensitive cells in 1 (of only 3 tested) dog. Dysfunction of lymphocytes per se (detected by a decreased blastogenesis in nonsuppressive normal canine and bovine sera) was detected in 3 dogs with demodicosis with pyoderma. The success of the treatment of demodectic mange or the bacterial skin infection did not correlate with the previous presence or absence of the blastogenesis suppressing SLIF. The treatment of pyoderma was less successful in dogs with an increase in blastogenesis of unstimulated cells in fresh normal canine serum over that in autologous serum. All 3 dogs with a detected dysfunction of their lymphocytes either died or were euthanatized as untreatable cases. It is concluded that the development of demodectic mange per se did not cause the appearance of the blastogenesis suppressing SLIF, which was primarily related to the appearance and extent of the secondary bacterial skin infection.     

Postnatal development of leukocyte subset composition and activity in dogs

The aim of the presentation is to summarise our data on the counts and activity of circulating canine leukocytes at birth and on their changes in the first 3 months of life. On day 1, neutrophil counts were almost three times higher than lymphocyte counts. During the first week of life, a decrease of neutrophil and an increase of lymphocyte counts, resulting in a predominance of lymphocytes, were observed. Neutrophil counts reached values comparable with those in adults in 1 month. Lymphocyte counts were higher than those in adults during the first 3 months. From birth to the age of 3 months, the phagocytic activity of neutrophils was nonsignificantly higher than in young adults. When compared with adults, the peripheral blood of new-born pups contained a lower proportion of T lymphocytes (detected by CD3 and CD5 markers), with a very low percentage of CD8(+) cells and a higher proportion of CD21(+) B lymphocytes. The counts of individual subsets levelled out during the first 3 months of life, although the proportion of CD21(+) B cells remained higher all the time. Lymphocytes of new-born pups were able to respond to nonspecific mitogen stimulation. Spontaneous proliferation in vitro was higher during the first week of life. Although in vitro stimulation of lymphocytes with Concanavalin A in some pups was comparable with that of adult dogs, mean activity was weaker. Pups with zero or very low levels of maternal antibodies were able to develop specific immune responses to a parvovirus antigen as early as at 2 weeks of age. On the basis of these data, we assume that pups are born with an immune system that can respond to external stimuli. Nevertheless its development continues in the postnatal period and some parameters differ from adult values for at least 3 months after birth.     

Effects of age, sex, and body size on serum concentrations of thyroid and adrenocortical hormones in dogs

Thyroxine (T4), 3,5,3'-triiodothyronine (T3), and cortisol frequently are quantified in canine serum or plasma samples to aid in the diagnosis of hypothyroidism, hypoadrenocorticism, and hyperadrenocorticism. Many laboratories have established reliable references values for concentrations of these hormones in blood of clinically normal animals. However, nonpathologic factors that affect thyroidal and adrenocortical secretion may lead to misinterpretation of test results when values for individual animals are compared with reference values. The objective of the study reported here was to identify effects of age, sex, and body size (ie, breed) on serum concentrations of T3, T4, and cortisol in dogs. Blood samples were collected from 1,074 healthy dogs, and serum concentrations of the iodothyronines and cortisol were evaluated for effects of breed/size, sex, and age. Mean (+/- SEM) serum concentration of T4 was greater in small (2.45 +/- 0.06 micrograms/dl)- than in medium (1.94 +/- 0.04 micrograms/dl)- or large (2.03 +/- 0.03 micrograms/dl)-breed dogs, the same in females (2.11 +/- 0.04 micrograms/dl) and males (2.08 +/- 0.04 micrograms/dl), and greater in nursing pups (3.04 +/- 0.05 micrograms/dl) than in weanling pups (1.94 +/- 0.05 micrograms/dl), rapidly growing dogs (1.95 +/- 0.04 micrograms/dl), and young adult (1.90 +/- 0.06 micrograms/dl), middle-aged adult (1.72 +/- 0.05 micrograms/dl), or old adult (1.50 +/- 0.05 micrograms/dl) dogs. Dogs greater than 6 years old had lower mean serum T4 concentration than did dogs of all other ages, except middle-aged adults. Mean serum T3 concentration in medium-sized dogs (1.00 +/- 0.01 ng/ml) was greater than that in small (0.90 +/- 0.01 ng/ml)- and large (0.88 +/- 0.01 ng/ml)-breed dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prognostic usefulness of blood leukocyte changes in canine parvoviral enteritis

BACKGROUND: Despite treatment, many dogs still die of complications related to canine parvoviral (CPV) enteritis. Effective prognostication would be beneficial in managing this disease. HYPOTHESIS: We hypothesize that the occurrence of leukocytopenias at admission and at 24 and 48 hours after admission, and changes in absolute leukocyte counts over time, could be used to predict outcome. ANIMALS: Sixty-two puppies with confirmed CPV. METHODS: A prospective study was performed. CBC was performed daily until discharge or death (in which case a postmortem examination was performed). RESULTS: Of the nonsurvivors (10/62; 16%), 9 died because of complications of the disease and 1 was euthanized because of a poor prognosis. There was a statistical significant difference in the occurrence of leukocytopenias between groups at 24 and 48 hours postadmission. The survivors showed a significant increase over time in certain leukocyte types (specifically lymphocytes) compared with values at admission. The positive predictive value for survivors was high. Nonsurvivors had marked thymic and lymphoid atrophy and marked bone marrow hypocellularity. CONCLUSION: An accurate prognosis could be obtained at 24 hours after admission by evaluating the change in total leukocyte, band neutrophil, lymphocyte, monocyte, and eosinophil counts.     

Haematological and biochemical changes in dogs naturally infected with Angiostrongylus vasorum before and after treatment

Haematological and biochemical parameters were studied prospectively in 48 dogs naturally infected with Angiostrongylus vasorum in a primary care setting. Samples for analysis were obtained when treatment was started and 42days afterwards. Prior to treatment, 21% of affected dogs exhibited eosinophilia, whereas increased total white blood cell (WBC) counts and neutrophilia were observed in only 4.2%. WBC counts and concentrations of neutrophils, eosinophils and monocytes decreased significantly from days 0 to 42, indicating that, even in dogs without elevated absolute blood values, a low grade inflammatory response may be present in dogs with A. vasorum infection. Biochemical changes (especially an increase in serum globulins and a decrease in serum fructosamine) were in agreement with the findings of other studies. The results show that the diagnosis of canine angiostrongylosis should not be excluded based on unremarkable haematological and blood biochemical parameters. They also support our recent finding that a low serum fructosamine concentration may be associated with infection with A. vasorum.     

Early-life longitudinal survey of peripheral blood lymphocyte subsets in Beagle dogs

A longitudinal study of peripheral blood lymphocyte subsets from 7 to 15 months of age was performed in Beagle dogs employing a multiparametric flow cytometry. The data were compared with data obtained from adult Beagle dogs that were housed in the same animal facilities and that were subjected to the same controls during the 34 weeks of the study. Absolute counts of total lymphocytes and CD3+ T, CD3+CD4+ Th and CD21+ B lymphocytes decreased during the entire 34 weeks period of the study in the young dogs group. The same was observed with regard to the percentage of CD3+CD4+ Th lymphocytes and the CD4/CD8 ratio, while the percentage of CD3+CD8+ Tc lymphocytes increased from 7 to 15 months of age. These age-related changes found in lymphocyte subsets distribution of young dogs led to level the absolute and relative values of adult dog lymphocytes. The observations of this longitudinal study illustrate the changes related to maturation of lymphocyte subsets that occur during early life in Beagle dogs.     

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND: Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. Objective: The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS: CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS: Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS: Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.     

Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.     

Serum biochemical and hematologic values of normal and Mycobacterium bovis-infected American bison

Hematologic and serum biochemical tests were used to monitor the health of 3 groups of bison in an experimental study of tuberculosis. Bison were randomly assigned to Mycobacterium bovis-infected, M. bovis-sensitized, and uninfected control groups. Hematologic measurements included total and differential leukocyte counts, hemoglobin (Hb), packed cell volume (PCV), fibrinogen, and plasma proteins. Biochemical tests included serum urea nitrogen, creatinine, aspartate amino transferase, sorbitol dehydrogenase, serum calcium, and serum phosphorus. There were no significant differences (P = 0.05) in any test values between groups of bison. The bison data were combined and compared to similar data of cattle. The mean values for PCV and Hb were higher than values (PCV 24-46%, Hb 8-15 g/dl) for cattle. Mycobacterium bovis-infected bison had a slight increase in the number of blood monocytes and lymphocytes when compared to the uninfected bison but were within the normal ranges for bison and cattle. Other hematologic parameters were within normal ranges reported for cattle. Creatinine levels in all bison were above the normal range (1.0-1.5 mg/dl) for cattle. Phosphorus levels for M. bovis-infected and M. bovis-sensitized bison exceeded the normal range (5.6-8.0 mg/dl) reported for cattle. The level for uninfected bison was near the upper limit of normal for cattle. Mean values for other serum biochemical tests were within the normal ranges reported for cattle.