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  • Author or Editor: Douglas W. Morck x
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Abstract

Objective—To examine the host response toward Porphyromonas levii,by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro.

Sample Population—Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer.

Procedure—Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P leviiin Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P levii following incubation with or without anti-P levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay.

Results—Chemotaxis toward P levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P levii with high-titer anti-P levii serum or anti-P levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only.

Conclusions and Clinical RelevancePorphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P levii foot rot lesions in cattle. (Am J Vet Res 2002;63:757–762)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7.

Sample Population—Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot).

Procedures—Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O2−and H2O2, respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining.

Results—All bacteria produced at least 3 of the 4 SCFAs. Production of both O2− and H2O2 was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O2− production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H2O2 production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5.

Conclusions and Clinical Relevance—Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of oral administration of tilmicosin in piglets experimentally infected with Actinobacillus pleuropneumoniae.

Animals—Forty 3-week-old specific-pathogen free piglets.

Procedure—Piglets were assigned to 1 of 4 groups as follows: 1) uninfected sham-treated control piglets; 2) infected untreated piglets that were intratracheally inoculated with 107 CFUs of A pleuropneumoniae; 3) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received tilmicosin in feed (400 ppm [µg/g]) for 7 days prior to inoculation; or 4) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received chlortetracycline (CTC) in feed (1,100 ppm [µg/g]) for 7 days prior to inoculation. Bronchoalveolar lavage (BAL) fluid and lung tissue specimens of piglets for each group were evaluated at 3 or 24 hours after inoculation. For each time point, 4 to 6 piglets/group were studied.

Results—Feeding of CTC and tilmicosin decreased bacterial load in lungs of infected piglets. Tilmicosin delivered in feed, but not CTC, enhanced apoptosis in porcine BAL fluid leukocytes. This was associated with a decrease in LTB4 concentrations in BAL fluid of tilmicosin-treated piglets, compared with untreated and CTC-treated piglets, and also with a significant decrease in the number of pulmonary lesions. Tilmicosin inhibited infection-induced increases in rectal temperatures, as measured in untreated and CTC-treated piglets. Pulmonary neutrophil infiltration and prostaglandin E2 concentrations in the BAL fluid were not significantly different among groups at any time.

Conclusions and Clinical Relevance—Oral administration of tilmicosin to infected piglets induces apoptosis in BAL fluid leukocytes and decreases BAL fluid LTB4 concentrations and inflammatory lung lesions. (Am J Vet Res 2005;66:100–107)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate immunomodulatory properties of all-trans retinoic acid and a fully oxidized β-carotene dietary product in calves with Mannheimia haemolytica–induced pneumonia.

Animals—Twenty-five 6- to 10-week-old male Holstein calves for experimental inoculations and three 8- to 30-week-old Angus heifers for blood donations.

Procedures—In vitro, neutrophils and monocyte-derived macrophages isolated from blood of healthy Angus heifers were treated with all-trans retinoic acid (1μM) or fully oxidized β-carotene (8.3 μg/mL) for various times and assessed for markers of cellular death, antimicrobial function, and production of proinflammatory leukotriene B4. Following 28 days of dietary supplementation with fully oxidized β-carotene, Holstein calves were experimentally inoculated with M haemolytica. Bronchoalveolar lavage fluid was collected at 3 and 24 hours after challenge inoculation and analyzed for markers of apoptosis.

Results—In vitro, all-trans retinoic acid and fully oxidized β-carotene induced cell-selective, caspase-3–dependent apoptosis in neutrophils, which subsequently enhanced efferocytosis in macrophages. Conversely, neither treatment altered phorbol 12-myristate 13-acetate–induced oxidative burst, phagocytosis of nonopsonized zymosan (complement or antibody independent), or M haemolytica–induced leukotriene B4 production in bovine neutrophils. In vivo, fully oxidized β-carotene enhanced leukocyte apoptosis in bronchoalveolar lavage fluid as well as subsequent efferocytosis by macrophages without altering numbers of circulating leukocytes.

Conclusions and Clinical Relevance—Neutrophil apoptosis and subsequent efferocytosis by macrophages are key mechanisms in the resolution of inflammation. Findings for the present study indicated that all-trans retinoic acid and fully oxidized β-carotene could be novel nutraceutical strategies that may confer anti-inflammatory benefits for cattle with respiratory tract disease.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae–induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs.

ANIMALS Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen–free pigs.

PROCEDURES Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis.

RESULTS In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A pleuropneumoniae– and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae–inoculated pigs.

CONCLUSIONS AND CLINICAL RELEVANCE Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.

Full access
in American Journal of Veterinary Research