Immunosuppression in dogs with pyoderma and/or demodicosis.

The occurrence of decreased activity of the immune system was studied in a group of 66 dogs with various combinations of pyoderma and demodicosis. Our complex examination of the dogs included the following: leukocyte count, differential count, phagocytosis, blastogenic lymphocyte transformation and quantitation of total serum immunoglobulins, lysozyme and haemolytic complement. Immunosuppression was found in 19 (28.8%) cases. Immunosuppression was rare in dogs with demodicosis and did not appear without a concurrent pyoderma. An increase in the neutrophil counts and total serum immunoglobulin levels significant was found in dogs with demodicosis combined with pyoderma. On the contrary, marked immunosuppression was detected in dogs with deep pyoderma. A considerable immunosuppression was present in 7 of 10 German shepherds dog pyoderma (GSP). Significant depressions were found in phagocyte activity and lymphocyte activity. Immunosuppression was observed in 4 of 9 dogs in other breeds with uncomplicated deep pyoderma. All groups of dogs with pyoderma showed a significant increase in total serum immunoglobulins. Conclusion from these findings is that deep pyoderma more than Demodicosis was concerned with immunosuppression. German shepherds with deep pyoderma had more expressed immunosuppression than other breeds.

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.

Immunodeficiency associated with lymphosarcoma in a horse.

Immune system dysfunction and immunoglobulin deficiency was diagnosed in a 2-year-old horse with disseminated lymphosarcoma. Prolonged (35 days) parenteral nutrition was delivered to support the horse during a period in which immune function studies could be performed. Correction of nutritional compromise by use of parenteral nutrition did not correct the immunoglobulin deficiency, and results of lymphocyte phenotype testing did not indicate abnormal proportions of leukocytes. Lymphoblast transformation studies were suggestive of a circulating immunosuppressive factor in the horse's serum. Normal cell function was detected when the cells were stimulated in precolostral equine serum.

Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.

Inhibition of lymphocyte blastogenesis by whey.

Bovine whey samples were evaluated by use of lymphocyte-transformation tests to determine their effect on lymphocyte blastogenesis. Whey samples from mammary glands with clinical mastitis strongly inhibited DNA synthesis and blastogenesis in lymphocytes stimulated with mitogens or dividing because of bovine leukemia virus infection. Whey samples from apparently healthy glands either did not inhibit lymphocyte DNA synthesis or inhibited it to a lesser degree than did whey from mastitic glands. Degree of inhibition was dose-dependent. The molecules causing inhibition were noncytotoxic and underwent minimal binding to the lymphocytes. Inhibitory molecules were susceptible to various proteolytic and glycolytic enzymes, indicating a glycoprotein-like structure. Whey inhibited incorporation of thymidine if it was in the cell cultures during the early stages of stimulation. Incubation of lymphocytes in whey that inhibited thymidine incorporation did not affect DNA synthesis in subsequent culturing of the same cells without whey. Degree of inhibition was affected by the method of whey preparation.

Lymphocyte blastogenesis inhibition by milk whey as an indicator of mastitis.

Sixty milk whey samples prepared from quarters of five cows with a history of mastitis were tested for their ability to inhibit DNA synthesis in mitogen-stimulated lymphocytes. The inhibitory activity was compared with milk SCC, electrical conductivity, pH, and the number of colony-forming bacteria in the milk. Milk whey contained factors that inhibited DNA synthesis in cultured lymphocytes. Inhibition of mitogen-induced DNA synthesis increased with the clinical severity of mastitis and with increased values of indirect indicators of mastitis. The increases in inhibition and electrical conductivity were delayed past the increases in SCC. Milk whey (10 microliters) from quarters with clinical mastitis and from quarters with SCC greater than 900,000 inhibited 96 to 100%, 84 to 100%, and 69 to 100% of DNA synthesis in 3-d cultures of lymphocytes stimulated with Concanavalin A, phytohemagglutinin P, and pokeweed mitogen, respectively. The numbers of colony-forming bacteria correlated least with the inhibitory activity.

Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus.

Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled (32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweek mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.

Cryptosporidiosis associated with immunosuppression attributable to distemper in a pup.

Cryptosporidiosis, giardiasis, trichomoniasis, and distemper were diagnosed in a 6-month-old female Siberian Husky pup. Poor growth rate, mucopurulent ocular and nasal discharges, and diarrhea were observed. Results of immunologic studies revealed decreased serum IgG concentration and undetectable serum IgA concentration. Cultured lymphocytes yielded a less-than-adequate response to mitogen stimulation. The serum also contained a factor that suppressed mitogen stimulation in control cultured lymphocytes.

Effect of serum on lymphocyte blastogenesis. 2. Characterization of disease-induced immunosuppressive factors by chromatography and molecular weight determination.

Numerous infectious and noninfectious diseases are associated with the appearance of suppressive serum lymphocyte immunoregulatory factors (SLIFs). The suppressive SLIFs in sera from clinically healthy dogs and from dogs with bacterial (staphylococcal, brucellar) or mycotic (blastomycotic) infections were further characterized by dialysis, fractionation by ultrafiltrations and HPLC (high performance liquid chromatography) sieving, by affinity chromatography on protein A-Sepharose columns, and by DEAE-cellulose ion exchange chromatography. Factors of various molecular weights and of various elution patterns from DEAE-cellulose and affinity chromatography columns were taking part in the suppressive action of the whole serum. The 'common' inhibitors present in all sera were in the molecular weight range of 28 to 35 Kd, whereas the disease-induced suppressive SLIFs were present in various molecular weight categories. 'Common' suppressor SLIFs and some SLIFs from dogs with staphylococcal infections were partially dialysable; suppressive SLIFs induced in dogs with generalized brucellosis and blastomycosis were not dialysable. Protein A bound suppressive SLIFs from two of three dogs with staphylococcal pyodermas. DEAE-cellulose chromatography gave variable elution patterns with different animal sera. It is concluded that various suppressive SLIFs contribute to the immunosuppressive effect of the whole serum and no disease-specific suppressive SLIF could be identified.

Monoclonal gammopathy associated with naturally occurring canine ehrlichiosis.

Clinical, hematologic, and immunologic findings for 14 dogs with Ehrlichia canis monoclonal gammopathy were studied retrospectively. Epistaxis, anemia, thrombocytopenia, hypoalbuminemia, hypergammaglobulinemia, and proteinuria were documented in the majority of these dogs. The serum protein electrophoresis pattern was characterized by a distinct narrow-base monoclonal spike, by a broad-base monoclonal spike, or by a monoclonal spike superimposed on a polyclonal gammopathy. The monoclonal spike disappeared following tetracycline treatment for ehrlichiosis. The long-term prognosis following treatment was generally good. The diagnostic features of monoclonal gammopathy due to myeloma were compared with those of E. canis monoclonal gammopathy. Owing to numerous similarities in clinical, hematologic, and immunologic findings, we conclude that an E. canis antibody titer should be determined in all dogs in which a diagnosis of benign monoclonal gammopathy is contemplated or definitive evidence of myeloma, leukemia, or macroglobulinemia is lacking.

Effect of serum sources and colostral whey on bovine semen quality and spermatozoa immunoglobulin G immunofluorescence.

Heifer, bull, fetal calf sera, and colostral whey were used to evaluate the influence of protein concentrations on percent progressive motility, head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence of bovine spermatozoa using ejaculates from 10 bulls. In the first experiment, 10% (vol/vol) addition of undiluted colostral whey resulted in the highest head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Ten percent (vol/vol) addition of whey diluted to a protein concentration equivalent to fetal calf serum produced significantly lower agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Fetal calf serum was unable to produce agglutination and immunoglobulin G immunofluorescence of bovine spermatozoa. Heifer and bull sera produced similar responses for all seminal measurements. In Experiment 2, unheated whey and heifer serum resulted in higher response for all variables than heat inactivated whey and heifer serum. Whey treatment produced greater spermatozoal motility, agglutination, acrosomal integrity; and immunoglobulin G immunofluorescence than treatment with heifer serum. Spermatozoal immunofluorescence indicated antibodies in normal whey, bull, and heifer serum bound to spermatozoal membranes at the acrosomal region. Colostral whey was an effective source of agglutinin factor. Normal unheated whey and heifer serum did not cause sperm damage or immobilization.

Ascaris suum infection in pigs sensitizes lymphocytes but suppresses their responsiveness to phytomitogens.

The effect of Ascaris suum infection and treatment with fenbendazole on the blastogenic response of peripheral blood lymphocytes to A. suum antigens and to three phytomitogens was assayed by the lymphocyte transformation technique. Repeated infections with A. suum led to the development of sensitized lymphocytes primarily responding to egg hatching fluid antigen. Treatment with fenbendazole decreased the number of specific sensitized lymphocytes, but favorably increased the resistance of pigs to reinfection. Immunity to reinfection did not correlate with the strength of the blastogenic response to A. suum antigens. Repeated infection with A. suum negatively affected the development of the blastogenic response to phytomitogens in the pigs, leading to a partial depression of the responsiveness of lymphocytes and to the partial suppression by serum. Responses to pokeweed mitogen were affected more than the responses to concanavalin A and phytohemagglutinin.

Immunomodulatory effect of serum on lymphocytes--consequences for the study of immunostimulants in vitro and in vivo.

The immunoregulatory effect of serum or plasma on the functions of body cells is often overlooked. Plasma is the natural environment for the cells of the immune system for at least a part of their life span and is the supplier of nutrients and regulatory molecules to the lymphoid tissues harboring immunocompetent cells. The plasma or serum immunoregulatory factors affect both in vivo and in vitro testing of immunostimulants. The serum immunoregulatory factors may be grouped into supporting factors, augmenting (co-mitogenic) factors, mitogenic factors, and suppressive factors. Some of them develop physiologically and appear or disappear during ontogeny, aging, pregnancy and other physiologic changes, others appear in connection with various diseases. The augmenting and mitogenic factors are mostly poorly defined. The natural and disease-induced immunosuppressive factors are a heterogeneous family of molecules ranging from immunoglobulins, through alphaglobulins and lipoproteins up to unidentified molecules of molecular weights from less than 6000 daltons to over 200,000 daltons. Proteins or polypeptides seem to play an important role in natural immunosuppressive factors, polysaccharides in disease-induced immunosuppressive factors. The presence of immunomodulatory plasma or serum factors affects the evaluation of immunostimulants in both in vivo and in vitro testing. All the cells, serum or plasma of experimental animals or donors should be pre-tested and carefully selected when immunostimulants are tested.