The effect of a commercial probiotic product on the milk quality of dairy cows.

Probiotics intended to improve plant health and productivity of pastures grazed by dairy cow are becoming commercially available in Australia. Great Land (GL; Terragen Biotech Pty Ltd., Coolum Beach, QLD, Australia) is one such biologic soil conditioner and spray-on probiotic with a label claim of "acting to improve plant health and productivity." The objective of this study was to quantify the effect of GL on the milk quality of cows grazing pasture top-dressed with GL. Lactating dairy cows of mixed age and breed (primarily Holstein-Friesian), in their second lactation or greater, and at least 80 d in milk were enrolled and randomly allocated into 1 of 2 study groups: a treatment cow group (n = 98; cows grazed pasture that was top-dressed with GL according to the product label) and a control cow group (n = 114; cows grazed untreated pasture). As required, both groups were supplemented at the same rate with a mixed ration during the grazing period. Composite milk samples were collected weekly from each cow during the study and analyzed to determine milk components. Milk volumes were recorded at each milking using the herd management software of the study farm. Mean differences in the milk component variables were compared using mixed-effects linear regression models. After controlling for the effect of days in milk, cow lactation, and time since a cow entered the study, the treatment cows produced an average of 1.21 L/cow per day more milk (95% confidence interval: 0.34-2.08 L/cow per day) and more milk protein (0.03 kg/d; 95% confidence interval: 0.01-0.05 kg/d) than the control cows. Pasture cover and pasture consumption did not differ between the GL-treated and the untreated study paddocks grazed by the treatment or control cows. A limited amount of published data have examined the effect of probiotic pasture treatment on the milk quality of dairy cows. This study suggests that application of such products may be beneficial. The mechanisms associated with this type of outcome remain to be investigated.

Hydration in non-suckling neonatal Brahman-cross calves.

OBJECTIVE: To identify measures that most closely relate to hydration in healthy Brahman-cross neonatal calves that experience milk deprivation. METHODS: In a dry tropical environment, eight neonatal Brahman-cross calves were prevented from suckling for 2-3 days during which measurements were performed twice daily. RESULTS: Mean body water, as estimated by the mean urea space, was 74±3% of body weight at full hydration. The mean decrease in hydration was 7.3±1.1% per day. The rate of decrease was more than three-fold higher during the day than at night. At an ambient temperature of 39°C, the decrease in hydration averaged 1.1% hourly. Measures that were most useful in predicting the degree of hydration in both simple and multiple-regression prediction models were body weight, hindleg length, girth, ambient and oral temperatures, eyelid tenting, alertness score and plasma sodium. These parameters are different to those recommended for assessing calves with diarrhoea. Single-measure predictions had a standard error of at least 5%, which reduced to 3-4% if multiple measures were used. CONCLUSION: We conclude that simple assessment of non-suckling Brahman-cross neonatal calves can estimate the severity of dehydration, but the estimates are imprecise. Dehydration in healthy neonatal calves that do not have access to milk can exceed 20% (>15% weight loss) in 1-3 days under tropical conditions and at this point some are unable to recover without clinical intervention.

Efficacy of difloxacin in calves experimentally infected with Mannheimia haemolytica.

OBJECTIVE: To determine the efficacy of difloxacin, a novel fluoroquinolone antibiotic, in calves experimentally infected with Mannheimia haemolytica (formerly Pasteurella haemolytica). ANIMALS: Seventy-two 3-month-old Holstein calves. PROCEDURES: Calves were inoculated with M haemolytica intratracheally; after they developed clinical signs of pneumonic pasteurellosis, they were randomly assigned to 1 of 6 groups (n = 12/group). Calves in each group were treated with 10% difloxacin (2.5 or 5 mg/kg of body weight), 5% difloxacin (2.5 or 5 mg/kg), enrofloxacin (5 mg/kg), or saline (0.9% NaCl) solution (control group), once daily for 5 days, and clinical signs were scored daily. On day 15, calves were euthanatized, and the percentage of diseased lung tissue was calculated. Swab specimens of the lungs were submitted for bacterial culture. RESULTS: Mortality rate and percentage of diseased lung tissue were significantly higher and cure rate and average daily gain were significantly lower for control calves, compared with calves in the treatment groups; however, no significant differences were found among treatment groups. Mannheimia haemolytica was isolated from the lungs of 10 control calves and from at least 2 calves in each of the treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that difloxacin and enrofloxacin were equally effective for treatment of calves with experimentally induced pneumonic pasteurellosis. However, treatment of infected calves with difloxacin or enrofloxacin may not eliminate the organism.

Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages.

OBJECTIVE: To investigate receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by bacterial lipopolysaccharide (LPS), using tissue factor (TF) expression as the measurable functional endpoint. SAMPLE POPULATION: Pulmonary AM harvested from 1- to 4-month-old male Holstein calves. PROCEDURE: Alveolar macrophages, acquired by use of volume-controlled bronchopulmonary lavage, were treated with CD14 monoclonal antibody (20 microg/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 microM chelerythrin, 100 microM H-7, or 50 nM staurosporin), then were stimulated with LPS alone (0.01, 0.10, 1.0, 10.0 microg/ml) or LPS (0.25, 0.5, 1.0 ng/ml) in combination with concentrated bovine serum fraction 2 (500 ng/ml). Tissue factor expression was quantified by use of a colorimetric assay. Changes in intracellular Ca2+ concentration and pH were monitored, using Ca2+- and pH-sensitive fluorescent dyes, with changes in fluorescent intensity after incubation with LPS measured by spectrophotometry. RESULTS: Treatment of AM with a CD14 monoclonal antibody caused profound inhibition of TF expression (P < 0.0001) after stimulation by LPS combined with bovine serum fraction 2. Pertussis toxin had a significant (P < 0.0319) inhibitory effect on TF expression when cells were stimulated by LPS alone. Treatment with all 3 PKC inhibitors caused marked reduction in TF expression of cells stimulated with LPS alone or with phorbol myristate acetate. Stimulation of cells by LPS failed to mobilize intracellular Ca2+ stores or to alter cytosolic pH. CONCLUSION: LPS combined with serum factors binds to CD14 on the surface of AM, and PKC is an important signaling kinase in the pathway utilized by LPS, resulting in enhanced TF expression; a pertussis toxin-sensitive G protein is involved in the signaling pathway utilized by LPS alone; and mobilization of Ca2+ does not have a role in the signal transduction pathway utilized by LPS nor does LPS affect cytosolic pH of AM.

Induction of procoagulant activity in virus infected bovine alveolar macrophages and the effect of lipopolysaccharide.

Three viruses known to be associated with the bovine respiratory disease complex were evaluated in vitro for potential impact upon the procoagulant activity (PCA) of bovine alveolar macrophages (bAM). Cultures of bAM were inoculated with bovine parainfluenza virus Type 3 (PI-3), cytopathic bovine viral diarrhea virus (cpBVDV), non-cytopathic BVDV (ncpBVDV), or bovine herpes virus Type 1 (BHV-1) and incubated for several time periods (24, 48, 72, 96 h). BAM were then exposed to E. coli lipopolysaccharide (LPS), or LPS with bovine serum. The amount of PCA expressed was quantified using a chromogenic assay. Viral inoculation increased bAM expression of PCA (P < 0.01). The increase in PCA expression was larger at higher rates of viral inoculation (P < 0.01). LPS enhanced PCA expression by bAM at low rates of viral inoculation (P < 0.01). The effect of LPS-serum treatment was greater than the LPS alone (P < 0.01). At high rates of viral inoculation, LPS had no enhancing effect on PCA expression. The effect of LPS on virus inoculated bAM varied with virus type, rate of inoculation, and duration of virus exposure (P < 0.01). The results suggest that these four viruses initiate the production of PCA by bAM independently of LPS. In the field situation, an initial viral infection may induce fibrin deposition in the pulmonary alveoli prior to the establishment of a secondary gram negative bacterial infection.

Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages.

Alveolar macrophages play a central role in host defense in the lower respiratory tract. Production of the reactive intermediate nitric oxide (NO), via expression of inducible nitric oxide synthase (iNOS) is an important microbicidal effector mechanism possessed by macrophages. In this study, cytokine regulation of NO production by bovine alveolar macrophages (bAM) was evaluated. Bovine alveolar macrophages were exposed to one or more of the following: recombinant human (rh) and recombinant bovine (rb) IFN gamma, rh- and rbIL-1 beta, rbGM-CSF, rhTNF alpha, rhIL-4, endotoxin (LPS), fetal bovine serum (FBS), mitogen-stimulated bovine splenic supernatant (SS), and purified human TGF beta-1. LPS alone, or in combination with SS, rbIFN gamma, or rbIL-1 beta stimulated production of NO in a time and dose dependent fashion. Recombinant bovine IFN gamma, rbIL-1 beta, and rhTNF alpha in combination produced maximal stimulation which was not further enhanced by LPS. Recombinant human IFN gamma, rhIL-1 beta, and rbGM-CSF had minimal effect either as single stimuli, or in combination with LPS, rbIFN gamma, rbIL-1 beta, or rhTNF alpha. Nitric oxide production was inhibited by rhIL-4, and the L-arginine analogue antagonists of iNOS, N-G-monomethyl-L-arginine (NGMMA) and aminoguanidine (AG). Purified human TGF beta-1 did not inhibit NO production. Messenger RNA for iNOS was maximally expressed by 8 h and remained detectable for at least 48 h. Expression of iNOS mRNA induced by cytokines and LPS varied with strength of the stimulus as determined by nitrite production in culture supernatant.

Attempt to pharmacologically modulate procoagulant activity of lipopolysaccharide-stimulated adherent bovine alveolar macrophages.

OBJECTIVE: To investigate the effects of anti-inflammatory drugs on lipopolysaccharide-induced procoagulant activity of bovine alveolar macrophages. DESIGN: Procoagulant activity was induced in bovine alveolar macrophages from 4 healthy Holstein calves aged 6 to 16 weeks by incubation with lipopolysaccharide. 3 anti-inflammatory drugs were used at 4 concentrations and 3 times to pretreat the alveolar macrophages. Results were analyzed to determine whether drug, concentration, or exposure period had a significant (P > 0.05) effect. PROCEDURE: Bovine alveolar macrophages, harvested by volume-controlled bronchoalveolar lavage, were pretreated for 30, 60, or 120 minutes with an anti-inflammatory compound (dexamethasone, flunixin meglumine, or phenylbutazone) at several concentrations ( 0, 1, 10, and 100 microM). Bovine alveolar macrophages were exposed to lipopolysaccharide (Escherichia coli O55:B5) in the presence and absence of fetal bovine serum for 4 hours. Procoagulant activity was measured, using a chromogenic assay. RESULTS: None of the drugs was associated with a modification of procoagulant activity expression. CONCLUSION: Use of these 3 anti-inflammatory drugs is unlikely to modify the extent of the fibrinous reaction commonly observed in cases of acute bovine respiratory tract disease complex. CLINICAL RELEVANCE: The alveolar macrophage has a key role in fibrin production. Assuming in vivo events mimic the in vitro model, is appears unlikely that administration of anti-inflammatory drugs will reduce the procoagulant activity of the bovine alveolar macrophages and the directly associated pulmonary fibrosis.

Bacterial lipopolysaccharide-stimulated nitric oxide generation is unrelated to concurrent cytotoxicity of bovine alveolar macrophages.

Nitric Oxide (NO) is a reactive metabolite produced by stimulated macrophages, and it has been demonstrated to exert cytotoxic actions on a number of microbes, parasites, and tumor cells. In addition, NO has been reported to have an autotoxic effect on murine macrophages, its site of synthesis. We have investigated the relationship of NO generation to cytotoxicity of bovine alveolar macrophages (AM) in vitro, and have also assessed the effects of several modulators of cellular function on this relationship. NO was generated in cultures of AM using sodium nitroprusside (SNP) and measured as [nitrite]. Cellular viability of AM reflected a strong, negative correlation with the concentration of NO/nitrite in supernatants (r = -0.987). Supernatants with nitrite concentrations in excess of 30 microMs were correlated with cytotoxicity. AM stimulated with the potent combination of endotoxin (Lipopolysaccharide, LPS; 10 ng/ml) and recombinant bovine IFN gamma (100 U/ml) also exhibited cytotoxicity over a 48-hour incubation period, and cells deteriorated to an average viability of 72.3% as compared to unstimulated control macrophages. In some cases the viability of macrophages was much lower. Even though LPS-mediated cytotoxicity occurred, the [nitrite] produced in supernatants during the 48-hour period (12.23 microMs) was well below the minimum concentration of SNP-generated NO required to induce cytotoxicity to macrophages. N(G)-monomethyl-L-arginine (N(G)MMA, 2 mM) is a competitive inhibitor of NO synthesis and was found to reduce nitrite concentrations from 12.23 microMs to 1.56 microMs in supernatants of LPS-stimulated AM, but this reduction did not promote increased viability of AM. Other modulators of cellular function including phenylbutazone (PBZ, 100 microMs), flunixin meglumine (FM, 100 microMs) and interleukin-4 (IL-4, 100 ng/ml) modestly inhibited synthesis of NO, but did not improve cellular viability. These results suggest that relatively high concentrations of exogenously-generated NO are toxic to AM in vitro, but the quantity of endogenously-generated NO synthesized by LPS-stimulated bovine AM is usually below the threshold for toxicity. Cytotoxicity occurs independently of NO synthesis, and factors other than NO are apparently responsible for LPS-related cytotoxicity to bovine macrophages.

Allergic rhinitis in a herd of cattle.

Allergic rhinitis was identified in a herd of Hereford cattle. Affected cattle had clinical signs of rhinitis (eg, nasal discharge, sneezing, nasal irritation, and nasal pruritus) and multiple small proliferative lesions in the nasal passages. Eosinophils were the predominant cell type in nasal discharges, and histologic examination of nasal mucosa biopsy specimens revealed chronic proliferative eosinophilic rhinitis. Results of CBC were normal; plasma fibrinogen concentrations were within reference limits. Results of intradermal allergen sensitivity testing and an ELISA for allergen-specific IgE only suggested an exaggerated IgE-mediated response to environmental allergens. Allergen-specific IgG may have acted as a competitive blocking antibody and limited clinical signs of disease in some cattle.

Bleeding diathesis associated with variant von Willebrand factor in a Simmental calf.

A 10-month-old Simmental heifer was examined because of a 10-day history of epistaxis and aural hematomas. Examination of the calf also revealed hemarthrosis. Initial laboratory data indicated that platelet count, platelet size, prothrombin time, and partial thromboplastin time were not different from a clinically normal (control) cow. Mucosal bleeding time was prolonged, and platelet adhesion to glass beads was less than expected. The clinical signs, prolonged bleeding time, and platelet adhesion defect were corrected by infusion of bovine plasma. Subsequent laboratory testing revealed that the affected calf had a truncated multimeric structure of von Willebrand factor (vWF), low vWF activity, and impaired platelet aggregation in response to adenosine diphosphate, but concentration of vWF was not different from that in clinically normal control animals. These data were consistent with a diagnosis of variant von Willebrand disease. The relationship of this disease to the thrombopathy of Simmental cattle is unclear.

Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.

Interaction of bovine respiratory syncytial virus with bovine alveolar macrophages in vivo: effects of virus infection upon selected cell functions.

The effect of bovine respiratory syncytial virus (BRSV) upon alveolar macrophage (AM) function was investigated using an in vivo calf inoculation model. Alveolar macrophages were collected sequentially from live calves at multiple time points during the 14 day period following viral inoculation. Alveolar macrophages from bronchoalveolar lavage fluids were purified by density gradient centrifugation (> 95% AM) prior to in vitro evaluation of cell functions. There were significant but variable and inconsistent differences in the functions of AM from the BRSV inoculated calves compared to the control calves. Fc-receptor mediated phagocytosis was either increased or unchanged by BRSV inoculation. Nonopsonized phagocytosis was decreased during the early postinoculation period and later increased. There was a variable effect on AM phagosome lysosome fusion with increased fusion activity on postinoculation days 2 through 5, 7 and 12 but reduced activity on days 6 and 10. The AM respiratory burst, as measured by nitroblue tetrazolium dye reduction, was essentially unaffected with a reduction in activity on day 10 only. In this model, BRSV inoculation of calves primarily resulted in an alteration of the membrane associated phagocytic functions of the alveolar macrophages (p < 0.05).

Lactose intolerance in a calf.

Lactose intolerance was identified as the cause of bovine neonatal diarrhea. Glucose and xylose oral absorptions were normal whereas lactose absorption was reduced relative to normal calves. Lactase deficiency is common in people but rarely reported in animals. The treatment of whole milk with lactase alleviated the diarrhea.

Tumor necrosis factor alpha and interleukin 1 alpha enhance lipopolysaccharide-mediated bovine endothelial cell injury.

Alveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such as Pasteurella haemolytica-induced pneumonia in cattle. Previous work has shown that AMs enhance P. haemolytica-mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 alpha (IL-1) or tumor necrosis factor alpha (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-1 receptor antagonist eliminated the IL-1 enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.

Alveolar macrophage and neutrophil interactions in Pasteurella haemolytica-induced endothelial cell injury.

Pasteurella haemolytica, the cause of fibrinous pleuropneumonia in cattle, produces extensive microvascular endothelial cell damage. This study investigated endothelial cell-leukocyte interactions by using a Millicell coculture assay system that modeled the bovine pulmonary alveolar defense system and showed that P. haemolytica-mediated endothelial cell damage was enhanced by the presence of alveolar macrophages, presumably due to soluble alveolar macrophage products. The alveolar macrophage-enhanced endothelial cell damage occurred regardless of the presence of anti-P. haemolytica immune serum; however, neutrophils and immune serum effectively prevented endothelial cell damage. These results suggest that alveolar macrophages are ineffective in controlling P. haemolytica growth and actually promote endothelial cell damage.

An in vitro model system to evaluate pulmonary macrophage, endothelial cell, and neutrophil interactions.

Pasteurella haemolytica, the cause of fibrinous pleuropneumonia in cattle, produces extensive microvascular endothelial cell (EC) damage. We have developed an in vitro model system to study the inflammatory process of this disease involving the interaction of pulmonary alveolar macrophages (AM), neutrophils (PMN), and EC. Bovine EC are grown to confluency in 24 well tissue culture plates. To mimic the vascular component, 10(6) PMN are later added to the EC monolayers. Bovine AM are plated onto millicell inserts and placed into the wells containing EC and PMN. The millicell insert serves as a semi-permeable barrier between AM and EC, allowing the exchange of only diffusible material. Preliminary work demonstrates that P. haemolytica stimulated AM resulted in EC damage presumably due to both soluble lipopolysaccharide and AM secreted products.