Mast cells in the bovine lower respiratory tract: morphology, density and distribution

The lower respiratory tract of six 8-day-old calves and six adult cows was fixed in either isotonic formal-acetic-acid or neutral buffered formalin in order to study the morphology, density and distribution of mast cells. They were found at all levels of the tract with the highest density in the major bronchi. Tissues from cows had significantly more mast cells than those from calves. There were significantly more mast cells detected in calf airways fixed with isotonic formal-acetic-acid compared with those seen in the same tissues fixed with neutral buffered formalin. Regardless of the age and fixation, mast cells were located predominantly in the alveolar septa and in the lamina propria of airways. They were also commonly encountered within the mucosal epithelium of the trachea. Ultrastructurally, mast cells in bovine airways and lung contained two types of intracytoplasmic granules as described in other species.     

Proliferative response of mammary gland mononuclear cells to recombinant bovine interleukin-2.

Methods of augmenting bovine mononuclear cell responsiveness during physiological transitions of the udder may enhance resistance of the mammary gland to intramammary infections. Interleukin-2 is required for proliferation of T-lymphocytes and may contribute to B-lymphocyte proliferation. Recombinant bovine interleukin-2 (rBoIL-2) was evaluated as a potential immunoenhancer of bovine mammary gland mononuclear cells. Bovine mononuclear cells were isolated from five primiparous Holstein cows at 14-18 and 28-32 days of involution and at 7-13 days prior to parturition. Bovine blood and mammary gland mononuclear cells were highly responsive to rBoIL-2. Response of mammary gland mononuclear cells to rBoIL-2 was comparable with response of blood mononuclear cells. These data suggest that rBoIL-2 may be an effective immunoenhancer of bovine mononuclear cells during the non-lactating and prepartum periods.     

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.     

Histopathological characteristics of Kupffer cells and ito cells in the porcine and bovine liver

We previously reported that no Kupffer cells reacted with the antibody against lysozyme, and Ito cells contained a large cytoplasmic vacuole in the feline liver. In this report, we further examined the characteristics of porcine and bovine hepatic non-parenchymal cells. In the liver of both animals, Kupffer cells were positive for lysozyme, and cytoplasmic vacuoles in Ito cells were small. The histopathological characteristics of porcine and bovine hepatic non-parenchymal cells were different from those of the feline liver.     

Comparison of the response of bovine and human neutrophils to various stimuli.

Elastase release, oxidant production and cytoplasmic Ca2+ fluxes by bovine and human neutrophils were compared using sensitive kinetic assays on a photon-counting spectrofluorometer. The stimulants used were phorbol myristate acetate (PMA), cytochalasin B, zymosan opsonized with bovine complement (bOZ) or human complement (hOZ), calcium ionophore, formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The respiratory burst of bovine and human neutrophils was stimulated by PMA and OZ but not by cytochalasin B, or calcium ionophore. Con A weakly stimulated this response in human neutrophils but not bovine. FMLP stimulated the respiratory burst of human but not bovine neutrophils. For evaluation of elastase release, human neutrophils were pretreated with cytochalasin B for 5 min and then stimulated. Cytochalasin B alone did not stimulate elastase release from human neutrophils. Phorbol myristate acetate, calcium ionophore, hOZ, FMLP and Con A did stimulate human neutrophils pretreated with cytochalasin B to release elastase. Human serum OZ was also able to stimulate elastase release from human neutrophils not pretreated with cytochalasin B. Some bovine neutrophils released elastase in response to cytochalasin B alone. Those bovine neutrophils that did not release elastase in response to cytochalasin B alone released elastase when stimulated with Con A or calcium ionophore after cytochalasin B pretreatment. Bovine neutrophils did not release elastase in response to FMLP or PMA with or without cytochalasin B pretreatment, but did release elastase in response to bOZ alone. Total elastase activity of bovine neutrophils was determined to be about 50 times less than that of human neutrophils. Intracellular calcium fluxes were stimulated in human neutrophils by calcium ionophore, FMLP, hOZ and Con A but not by PMA or cytochalasin B. Bovine neutrophil calcium fluxes were stimulated by calcium ionophore, Con A and bOZ; cytochalasin B also stimulated bovine neutrophils to increase cytoplasmic calcium concentration. Cytoplasmic calcium fluxes were not stimulated in bovine neutrophils by PMA or FMLP. In summary, human and bovine neutrophils respond similarly to calcium ionophore and OZ, but differently to PMA, cytochalasin B, Con A and FMLP.     

Leukocyte response of bovine mammary gland to injection of killed cells and cell walls of Staphylococcus aureus.

Injection of mammary glands of cows with heat-killed staphylococcal cells, staphylococcal cell walls, or distilled water induced leukocytosis. The magnitude of leukocytic response to staphylococcal antigens in cows of each treatment group depended on the extent of previous experience with staphylococcal cell materials. The 2nd intramammary injection produced greater reactions than did the 1st, and the 1st injection in cows previously parenterally vaccinated with killed cells in oil-water adjuvant produced responses comparable with those elicited by the 2nd intramammary injection in nonvaccinated cows. The implications that these changes have toward understanding the pathogenesis of, and immunity to, staphylococcal mastitis were discussed.     

Effect of Ostertagia ostertagi secretions and various putative secretagogues and inhibitors on aminopyrine accumulation in dispersed bovine abomasal gland cells

Aminopyrine accumulation in suspensions of isolated gastric glands was used to determine the effect of Ostertagia ostertagi secretions and putative secretagogues and inhibitors on abomasal parietal cells. Parasite secretions did not affect acid production nor did histamine. Dibutyryl cyclic adenosine monophosphate (cAMP) and pentagastrin significantly increased aminopyrine accumulation by gastric glands and cimetidine, omeprazole and thiocyanate significantly decreased aminopyrine accumulation confirming their roles as stimulators and inhibitors of gastric acid production, respectively.     

Hematology,morphology, cytochemical staining,and ultrastuctural characteristics of blood cells in king cobras (Ophiophagus hannah)

Background — King cobras (Ophiophagus hannah) have been captive‐bred at Queen Saovabha Memorial Institute since 1996 to supply venom for antivenom production. Hematologic tests would be useful for evaluating the health of the snakes, however, basic hematologic data and morphology have not been described for this species. Objective — The purpose of this study was to determine basic hematologic values and evaluate light microscopic, cytochemical, and electron microscopic characteristics of king cobra blood cells. Methods — Blood samples from 13 wild‐caught and 15 captive‐bred king cobras were collected into EDTA from the ventral caudal vein. A CBC was done using standard methods. Significant differences between groups were determined using t‐tests. Cytochemical stains (periodic acid‐Schiff [PAS], Sudan black B [SBB], a‐naphthyl acetate esterase [ANAE], acid phosphatase [AcP], and p‐glucuronidase [p‐glu]), and scanning and transmission electron microscopy were done using standard techniques. Results — Eighteen snakes (64.3%) were positive for Hepatozoon infection. Hepatozoon organisms were detected nearly twice as frequently in wild‐caught (11/13) as in captive‐bred (7/15) snakes.Total WBC, azurophil, and lymphocyte counts were higher and fib‐rinogen concentration was lower in Hepatozoon‐positive snakes. Captive‐bred snakes had higher RBC values, lower azurophil, het‐erophil and punctate reticulocyte percentages, and higher lymphocyte numbers compared with wild‐caught snakes. Lymphocytes were the most commonly observed WBCs, and stained positive with PAS, ANAE, AcP, and (3–glu. Azurophil granules stained positive with SBB, PAS, and ANAE. Heterophils were the largest WBCs; their granules stained with SBB, ANAE, and (3–glu. Basophil granules stained with PAS, SBB, ANAE, and (3–glu. Thrombocytes were strongly positive with PAS. Transmission electron microscopic examination revealed organelles within all WBCs except eosinophils and revealed the gamonts of Hepatozoon sp in RBCs and azurophils. Conclusions — These results provide comparative hematologic data and a guide for identification of blood cells in wild‐caught and captive‐bred king cobra snakes. Hepatozoon infection was relatively common, but was not associated with severe hematologic abnormalities.     

Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi)

The object of this study was to examine the erythrocytes, leukocytes and thrombocytes of the giant lizard of El Hierro (Gallotia simonyi) by light and electron (TEM) microscopy, and cytochemical staining. Smears were prepared from blood from the ventral coccygeal vein of 10 healthy adult lizards (five males and five females) from the Giant Lizard of El Hierro Reproduction and Research Centre, Canary Islands, Spain. The cytochemical stains used were: benzidine peroxidase (BP), chloroacetate esterase (CAE), alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), periodic acid-Schiff (PAS), toluidine blue (TB) and May-Grünwald-Giemsa (MGG). Electron microscopy was also performed on all samples. Heterophils had granules that were heterogeneous in both size and electron density, and stained with BP, PAS and ANAE. Eosinophil granules were homogeneously electron-dense and stained for AP, CAE and ANAE. Basophils had both highly and moderately electron-dense granules, and stained with TB and ANAE. Azurophil granules were of low electron-density and stained for AP, CAE and ANAE. Azurophil cytoplasm was vacuolated on TEM. The cytoplasm of lymphocytes contained many ribosomes and was positive for AP. Monocytes had a large nucleus and a vacuolated cytoplasm but did not stain by any of the cytochemical methods used. Thrombocytes had a relatively large nucleus but little cytoplasm; they did not stain cytochemically. The blood cells of the giant lizards of El Hierro differ from those of other members of the Order Squamata both morphologically and cytochemically. The variation in cytochemical responses in the blood of reptiles makes it necessary to study species individually if meaningful clinical decisions are to be made.     

Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

Morphologic, cytochemical staining, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus)

OBJECTIVE: To evaluate light microscopic, cytochemical, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes. ANIMALS: 10 healthy snakes. PROCEDURE: Various stains, including Wright-Giemsa, benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff with diastase, and toluidine blue, were used to stain leukocytes differentially on multiple blood smears. Electron microscopy also was performed. RESULTS: Lymphocytes were the most commonly observed leukocyte and could be distinguished from thrombocytes, using periodic acid-Schiff stain with diastase. Azurophils also were commonly observed; their granules stained with peroxidase. Eosinophils were not identified; however, 2 morphologic variations of heterophils were seen in the blood of all snakes and were considered the same cell type at different stages of cytoplasmic granule development. Heterophil granules were better preserved, using a one-step Wright-Giemsa method that did not require alcohol fixation prior to staining. Degranulated heterophils were observed in all preparations. CONCLUSIONS: Most leukocytes of eastern diamondback rattlesnakes can be identified easily on Wright-Giemsa-stained preparations. However, hematologic stains that do not require alcohol fixing prior to staining may be preferred for leukocyte evaluation in certain reptiles. A limited degree of heterophil maturation may continue in the blood of healthy snakes. This, along with degranulation of heterophils, may result in a variable staining pattern in this cell type, regardless of the stain used. CLINICAL RELEVANCE: Results provide baseline data for use in hematologic testing in diagnosis of disease and monitoring of treatment of sick or injured snakes.     

Effects of active immunization against GnRH on LH, FSH and prolactin storage, secretion and response to their secretagogues in pony geldings

Six pony geldings were actively immunized against GnRH conjugated to bovine serum albumin (BSA) to study 1) the relative dependency of LH and FSH storage, secretion and response to GnRH analog on GnRH bioavailability and 2) the effects of reduced GnRH bioavailability on GnRH storage in the hypothalamus. Five geldings were immunized against BSA. Geldings were immunized in December and 4, 8, 14, 20, 26 and 32 wk later. Ponies immunized against GnRH had increased (P less than .01) GnRH binding in plasma within 6 wk. By June, plasma concentrations of LH and FSH in ponies immunized against GnRH had decreased (P less than .02) by 86 and 59%, respectively, relative to ponies immunized against BSA. The LH response to an injection of GnRH analog, which did not bind to anti-GnRH antibodies, was reduced (P less than .005) by 90% in ponies immunized against GnRH relative to ponies immunized against BSA. In contrast, the FSH response to GnRH analog was similar (P greater than .1) for both groups. Immunization against GnRH reduced (P less than .05) weight of the anterior pituitary (AP) by 31%, LH content in AP by 91% and FSH content in AP by 55% relative to ponies immunized against BSA. There was no effect of GnRH immunization on prolactin characteristics or on GnRH concentrations in the median eminence, preoptic area or body of the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular interactions regulating the in vitro response of bovine lymphocytes to ovalbumin.

We examined the contribution of MHC class II-restricted T cells (CD4+), MHC class I-restricted T cells (CD8+), gamma/delta T cell receptor (TCR)+ T cells, B cells and macrophages to the development and control of in vitro proliferative responses of bovine lymphocytes to ovalbumin (OA). Cell populations for in vitro assay were obtained from peripheral blood (peripheral blood leukocytes, PBL) of OA-primed cattle. Specific cell populations were depleted or purified from PBL by staining with monoclonal antibodies (MAbs) against the appropriate differentiation antigens and sorting on a Fluorescence Activated Cell Sorter (FACS). OA-specific in vitro responses of in vivo primed PBL were dependent on the presence of CD4+ T cells. Their presence could not be replaced by the inclusion of T cell growth factor (TCGF) in the culture system, indicating that CD4+ T cells probably actively proliferate in response to antigenic stimulation. Bovine CD8+ T cells and gamma/delta TCR+ T cells appeared to exert a suppressive effect on proliferative responses. No proliferation was observed in PBL after the depletion of MHC class II+ cells. In this case, the response could be restored by the addition of macrophages or LPS-activated B cells to the MHC class II- population.     

The immune response to bovine viral diarrhea virus: a constantly changing picture.

Bovine viral diarrhea virus (BVDV) is one of the major immuno-suppressive viruses of cattle. The effect on the innate and acquired immune system is unique and results in dramatic immune dysfunction. BVDV infection also has the ability to cause persistent infection (PI) in the developing fetus. This Pl syndrome creates a requirement for high levels of BVDV immunity from vaccines to prevent these infections. BVDV vaccines and their future development continue to be an enigma in the control of BVDV.