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Effects of melengestrol acetate on the inflammatory response in heifers challenged with Mannheimia haemolytica¹

M. E. Corrigan^*, J. S. Drouillard^*,2, M. F. Spire, D. A. Mosier, J. E. Minton^*, J. J. Higgins, E. R. Loe^*, B. E. Depenbusch^* and J. T. Fox

^* Department of Animal Sciences and Industry, and Department of Diagnostic Medicine and Pathobiology, and Department of Statistics, Kansas State University, Manhattan 66506

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Previous research from our laboratory has indicated that melengestrol acetate (MGA) added to the diet during the first 35 d after arrival in the feedlot improves growth rates and tends to reduce chronic respiratory disease in heifers naturally challenged with bovine respiratory disease. The current study was conducted to provide further insight into the possible immunomodulatory effects of MGA. Crossbred heifers (n = 48; 232 ± 5.5 kg of BW) were used in a randomized complete block design to determine the effects of MGA on lung pathology and markers of inflammation after Mannheimia haemolytica challenge. On d 0, cattle were blocked by BW and randomly assigned, within block, to diets (54% concentrate) that provided 0 or 0.5 mg of MGA per heifer daily for the duration of the experiment. Inoculum containing from 1.3 x 10⁹ to 1.7 x 10⁹ cfu of M. haemolytica (20 mL) was instilled at the bifurcation of the trachea on d 14. Blood samples were collected, clinical observations were made, and rectal temperatures were recorded for each animal at 0, 12, 24, 48, 72, 96, 120, and 138 h after inoculation. Heifers fed MGA had greater circulating concentrations of eosinophils and postchallenge concentrations of segmented neutrophils and white blood cells (P < 0.01) than controls, as well as elevated plasma protein, serum haptoglobin, and fibrinogen after M. haemolytica challenge (P < 0.01). Heifers fed MGA had lower plasma glucose (P < 0.01), greater plasma urea N (P = 0.02), and elevated respiratory indices (P < 0.01) compared with controls. Necropsies performed on d 6 after inoculation suggested that M. haemolytica challenge was relatively mild, because lesions were confined to a small portion of the lungs. On a 0 to 100 scale, average lung lesion scores were 3 and 1 for MGA-fed and control groups, respectively (P < 0.06). Heifers fed MGA before mild M. haemolytica challenge were more susceptible to infection, as evidenced by a greater number of heifers fed MGA exhibiting pulmonary lesions 138 h after inoculation than controls (14 out of 23 vs. 6 out of 24 for MGA and controls, respectively; P < 0.02).

Key Words: heifer • Mannheimia haemolytica • melengestrol acetate

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Despite widespread use of antibiotics and vaccines, bovine respiratory disease (BRD) continues to be the most economically important disease affecting feedlot cattle. Losses associated with BRD are nearly $1 billion annually (Griffin, 1997), and Edwards (1996) reported that approximately 75% of morbidities and over 50% of mortalities in feedlot cattle are attributable to BRD.

The most common bacterial agent isolated from pulmonary tissues of pneumonic cattle is Mannheimia haemolytica (Rehmtulla and Thomson, 1981), which possesses several virulence factors, including leukotoxin and endotoxin [lipopolysaccharides (LPS); Confer et al., 1990]. Damage to endothelial cells from M. haemolytica LPS can occur either directly (Breider et al., 1990) or indirectly (Sharma et al., 1992) by stimulating alveolar macrophages. Leukotoxin and LPS from M. haemolytica have been shown to enhance the production of inflammatory cytokines like tumor necrosis factor- (TNF-) and IL-1 in alveolar macrophages (Yoo et al., 1995; Morsey et al., 1999).

Previous research from our laboratory has indicated that melengestrol acetate (MGA), a synthetic progestin commonly used to suppress estrus in feedlot cattle, improves growth rates and tends to reduce chronic sickness in prepubertal heifers naturally challenged with severe BRD when fed during the first 35 d after arrival in the feedlot (Sulpizio et al., 2003). Progesterone has been shown to have antiinflammatory properties in other species, including reduced TNF- production by LPS-activated mouse macrophages (Miller and Hunt, 1998). We hypothesized that the beneficial effects of MGA may have been due in part to antiinflammatory properties of MGA.

The study presented herein was conducted to evaluate the effects of MGA on biological markers of inflammation and lung pathology in heifers experimentally infected with M. haemolytica. Furthermore, several markers of the innate immune response to M. haemolytica were measured in an effort to better understand the response to the pathogen.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and Experimental Design

Procedures for this study were approved by the Kansas State University Institutional Animal Care and Use Committee. Forty-eight, medium-framed crossbred heifers (232 ± 5.5 kg of BW) were purchased from a local sale barn and used in a randomized complete block design to determine the effects of MGA on the inflammatory response in heifers after M. haemolytica challenge. Heifers received no vaccinations or antimicrobial treatments after arrival. Due to labor and facility restrictions, the experiment was conducted in 6 blocks consisting of 6 to 10 heifers each (with an equal number of animals from each treatment represented in each block). The beginning BW of the blocks were 220 ± 13, 220 ± 15, 223 ± 10.2, 233 ± 6.8, 242 ± 17.5, and 250 ± 13.7 kg.

Heifers were held at the facility for a period of 1 to 5 wk before being enrolled in the experiment to assess health or temperament issues that would make them unfit for study. Any heifers showing signs of illness, including rapid or labored respiration, nasal or ocular discharge, anorexia, or depression, were not used in the experiment. On d 0 of the experiment, heifers were individually weighed and assigned to blocks by BW, with an equal number of heifers assigned to each treatment within each block. The heifers were randomly assigned within block to either 0 or 0.5 mg of MGA per heifer daily. Diets consisted of 45.5% steam-flaked corn, 44.9% alfalfa hay, 5.1% corn steep liquor, and 4.5% of a protein, vitamin, and trace mineral supplement (DM basis). The MGA supplement was top-dressed onto each the ration of each heifer at a rate of 113 g/d, which provided 0 or 0.5 mg of MGA per heifer daily. The top-dressed supplement was then mixed into the ration by hand to ensure uniformity.

The heifers were then randomly assigned to individual pens (1.5 x 3.7 m) in a metal barn with slatted concrete floors. Diets were fed once daily at approximately 1100 in amounts that led to only traces being present at the next feeding (average DMI = 5.8 kg/d). Each day before feed delivery, unconsumed feed was collected, weighed, and analyzed for DM content. Refused feed was subtracted from the feed offered, and these values were used to determine daily feed intake and G:F.

Throughout the experiment, the heifers were evaluated for clinical signs of BRD, and rectal temperature was recorded once daily at approximately 0930. On d 14 of the experiment, all heifers were weighed, and a blood sample was taken via jugular venipuncture. Next, a 20-mL dose containing 1.3 to 1.7 x 10⁹ cfu (heifers in each block received the same number of cfu) of M. haemolytica was inoculated at the level of the bifurcation of the trachea of each heifer. The initial dose of 1.3 x 10⁹ was used in the first block and was increased throughout the study, because the desired severity of the challenge was not met in the early blocks. Animals were observed for clinical signs of BRD at 0, 12, 24, 48, 72, 96, 120, and 138 h after inoculation. At each observation point, the rectal temperature of the heifer was measured, and a blood sample was drawn. Before the 138-h sampling, each heifer was individually weighed. After the final blood sample, heifers were euthanized and immediately transported to the Kansas State University College of Veterinary Medicine Diagnostic Laboratory, where a postmortem examination was performed.

The lungs of each animal were harvested and observed for gross signs of active pneumonia, as determined by D.A. Mosier, who is a veterinary pathologist. Each animal was given a lung lesion score based on the percentage of each lung lobe affected by active lesions, using the formula described by Jericho and Langford (1982): total calculated lung lesion score = [(left cranial % x 0.05) + (left caudal cranial % x 0.06) + (left caudal % x 0.32) + (right cranial % x 0.06) + (right caudal cranial % x 0.05) + (right middle % x 0.07) + (right caudal % x 0.35) + (accessory % x 0.04)]. One animal in the MGA treatment group died between 48 and 72 h after inoculation. The cause of death was determined to be pneumonia, and the lesions were bifocal, affecting approximately 50% of the lung tissue, and thus were not consistent with lesions observed in other animals experimentally infected. We presume that the lesions observed in this animal were likely due to a preexisting condition, thus data from this animal were excluded from the statistical analyses.

Quantification of Clinical Observations

All clinical observations were made by an animal care manager who was blinded to treatments. Clinical scores were assigned using the following criteria: respiratory index: 0 = normal; 0.5 = shallow; 1 = labored; 2 = dyspnea; 3 = severe distress; activity level: 0 = normal; 0.5 = stiff gait; 1 = reluctant to move; 2 = down, able to rise; 3 = down, not able to rise; hydration index: 0 = normal; 0.5 = 50 to 75% of normal; 1 = less than 50% of normal; 2 = severe dehydration; appetite index: 0 = normal; 0.5 = 50 to 75% of normal; 1 = less than 50% of normal; 2 = no appreciable feed intake; fecal score: 0 = normal; 0.5 = loose; 1 = watery; 2 = blood and mucous; nasal discharge: 0 = none; 0.5 = slight nasal discharge; 1 = severe nasal discharge; and ocular discharge: 0 = none; 0.5 = slight ocular discharge; 1 = severe ocular discharge.

Inoculum Preparation and Challenge Procedure

Mannheimia haemolytica inoculum was prepared as described by Mosier et al. (1995). Briefly, M. haemolytica was grown on brain-heart infusion agar containing 5% bovine blood for 18 h at 37°C in 7% CO₂. Colonies were inoculated into brain-heart infusion agar and incubated for 6 h at 37°C in a rotary shaker bath. Broth tubes were then centrifuged at 3,000 x g for 30 min at 5°C. The pellet was then resuspended in PBS solution for a final concentration of 1.3 to 1.7 x 10⁹ cfu/20-mL dose. After preparation, the inoculum was placed on ice in a dark cooler and transported approximately 1.5 km from the Kansas State University College of Veterinary Medicine to the Beef Cattle Research Center, where it was immediately used for inoculation. For challenge exposure, catheters were fashioned from a 2.5-m length of 3.2-mm (i.d.) polyethylene tubing (489 polyethylene tubing, Nalge Co., Rochester, NY) with a 3-cm piece of 4.8-mm (i.d.) polyvinyl chloride tubing (Nalgene 180 clear PVC tubing, Nalge Co.) attached to the end. Catheters were gas-sterilized before use. Heifers were restrained in a manual squeeze chute with a head gate. The head was tied using halter ropes so that it faced upward, and the catheter was passed to the bifurcation of the trachea.

Blood Sample Collection

Blood samples were taken via jugular venipuncture and placed in evacuated tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Blood for plasma triglycerides, plasma urea N, plasma glucose, and plasma lactate quantification was placed in tubes containing Na heparin as an anticoagulant and centrifuged for 15 min at 2,000 x g using a Centra GP8R centrifuge (IEC, Needham Heights, MA) equipped with an IEC 228 rotor at 13°C. Blood for serum insulin, cortisol, NEFA, haptoglobin, and TNF- quantification was placed in tubes lacking anticoagulant, allowed to clot at room temperature for 2 to 3 h, and centrifuged as above. Whole blood for hematological profiles, blood cell counts, plasma fibrinogen, and hematocrit determination was placed in tubes containing EDTA as an anticoagulant. Blood for plasma protein determination was placed in tubes containing EDTA as an anticoagulant and centrifuged for 5 min at 13,460 x g using an IEC MB microhematocrit centrifuge equipped with an IEC 275 microhematocrit rotor.

Blood Analyses

Plasma triglyceride concentrations were determined by colorimetry using a commercially available kit (TR0100, Sigma, St. Louis, MO). Colorimetric determinations of plasma urea N (Marsh et al., 1965) concentrations were made using an autoanalyzer (Analyzer II, Technicon Industrial Systems, Buffalo Grove, IL). Concentrations of serum NEFA were determined colorimetrically using a commercially available kit (NEFA-C kit, ACS-ACOD method, Wako Chemicals US, Richmond, VA), which was modified as previously described by Eisemann et al. (1988). Plasma glucose and plasma lactate were measured using a glucose-lactate autoanalyzer (2300 Stat Plus, YSI Inc., Yellow Springs, OH). Serum haptoglobin was determined photometrically as described by Makimura and Suzuki (1982). Plasma protein and fibrinogen were measured with a refractometer using methods described by Jain (1986). Red blood cell (RBC) counts, white blood cell (WBC) counts, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration, and red blood cell distribution width were measured using a Cell-Dyn 3700 system (Abbott Laboratories, Abbott Park, IL). Differentiated leukocyte counts were performed on smear slides stained with a modified Wright stain (Sheehan and Hrapchak, 1980).

Commercially available RIA kits were validated to determine serum cortisol (Coat-A-Count cortisol, Diagnostic Products Corp., Los Angeles, CA) and serum insulin concentrations (Insulin RIA DSL-1600, Diagnostic Systems Laboratory Inc., Webster, TX). Samples were analyzed in 4 assays. For cortisol, bovine serum samples were diluted 1.25- and 1.67-fold, and the concentrations estimated by the assay averaged 111.1% of the expected concentrations when corrected for dilution. Quantitative addition of cortisol (Diagnostic Products Corp.) to bovine serum samples was measured at 105.8% of the expected concentrations. The calculated sensitivities (2 SD from the tracer binding in the zero tubes) of the assays used for this study averaged 0.98 ng/mL. The intra- and interassay CV were 9.3 and 11.4%, respectively. The insulin assay was similarly validated for bovine serum. Diluted samples (1.33-, 1.50-, and 1.67-fold) were measured in the assay at 92.7% of the expected value when corrected for dilution. Addition of bovine insulin (Diagnostic Systems Laboratory Inc.) into bovine serum samples yielded measured values of 104.2% of the expected concentration. The calculated sensitivities of the assays used for this study averaged 0.008 ng/mL. The intra- and interassay CV were 4.3 and 8.0%, respectively.

Serum TNF- was measured using a bovine-specific ELISA. Recombinant bovine TNF- (bTNF-) was produced commercially, with initial sequence selection and analysis assistance from Brad Johnson (Kansas State University). A 10 ng/mL aliquot of the recombinant, commercially produced protein was sent to an independent laboratory and was determined to have biological activity in the WEHI 13Var-164-based assay, and its concentration in that assay was confirmed to be 10 ng/ mL. This recombinant bTNF- was used as the standard in the assay and also for conjugation to keyhole limpet hemocyanin as antigen for immunization and antibody production. Polyclonal antisera were generated in rabbits against the bTNF--keyhole limpet hemocyanin conjugate and pooled. The antisera were then affinity-purifed against the recombinant antigen. These affinity-purified antisera were used as both the capture and biotinylated detection antibodies. The standard curve for the ELISA ranged from 78.1 pg/mL to 10 ng/ mL. Quantitative recovery of bTNF- in bovine serum samples to which recombinant bTNF- had been added (the assay measured 97.1 ± 3.1% of the expected bTNF-) and quantitative determination of TNF- in samples diluted 1.33- and 2-fold were used as variables to validate the ELISA (the assay measured 93.8 ± 10.2% of the undiluted sample). The intra- and interassay CV for samples quantified in 6 assays were 3.9 and 7.9%, respectively. The sensitivity of the assay was 0.08 ng/ mL.

Statistical Analyses

Blood parameters, observational scores, and temperature were analyzed as repeated measures (randomized complete block design) using the MIXED procedure (SAS Inst. Inc., Cary, NC). The model effects included treatment, sampling time, and treatment x sampling time. The random effect was block, and an unstructured variance-covariance matrix was assumed for repeated measurements. Average percentage of lung lesion scores and performance data were analyzed with PROC MIXED of SAS as a randomized complete block design. The model effect was treatment, and the random effect was block. Prevalence of lung lesions between treatments was analyzed using Fisher’s exact test with PROC FREQ of SAS.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Clinical Scores and Rectal Temperatures

Total observational scores and rectal temperatures are presented in Table 1. Data for activity level and fecal score did not meet the criteria for a repeated measures analysis in SAS. This was likely due to a high number of null values recorded for these 2 variables. Consequently, data for these variables were excluded from the final analysis. There were no effects of treatment, sampling time, or interaction between treatment and sampling time on appetite index, nasal discharge, or ocular discharge (P > 0.15). There were effects of treatment and sampling time on respiratory score and total observational score (P < 0.01), with heifers fed MGA having greater values for both variables. Respiratory scores were greater (P < 0.01) than baseline values 12 h after inoculation, whereas total observational scores were greater (P < 0.05) than baseline values 12 and 48 h after inoculation. Rectal temperature was not different between treatments (P = 0.95), but there was an effect of sampling time (P < 0.01) with an increase (P < 0.01) in rectal temperature 12 h after inoculation that returned to baseline (P = 0.15) 24 h after inoculation. Two heifers, one from each treatment, did not experience an elevation in rectal temperature 12 h after inoculation.

Red blood cell concentration and parameters are presented in Table 2. There were no differences between treatments in RBC concentration (P = 0.52), hemoglobin (P = 0.21), hematocrit (P = 0.14), MCH concentration (P = 0.72), or RBC distribution width (P = 0.13). There was an effect of sampling time on RBC concentration, hemoglobin, and hematocrit, with all 3 variables having lower (P < 0.01) values within 24 h after challenge compared with prechallenge concentrations. These values remained lower (P < 0.01) throughout the postchallenge period. Heifers fed MGA had greater MCV (P < 0.01) and MCH (P = 0.01) than controls, but there were no effects of sampling time (P = 1.00) on either variable.

White blood cell concentrations are presented in Table 3. Heifers fed MGA had greater circulating concentrations of WBC (P < 0.01), segmented neutrophils (P < 0.01), and eosinophils (P < 0.01) than controls, but there was no difference between treatments in circulating band neutrophil (P = 0.32), lymphocyte (P = 0.78), monocyte (P = 0.38), or basophil (P = 0.15) concentrations. Concentrations of WBC, segmented neutrophils, band neutrophils, monocytes, and basophils all changed from baseline concentrations in the postchallenge period (P < 0.05). Concentrations of WBC and segmented neutrophils were elevated (P < 0.01) 12 and 24 h after inoculation but returned to baseline concentrations (P > 0.05) by 48 h after inoculation. Concentrations of band neutrophils were also elevated (P < 0.01) 12 h after inoculation but returned to baseline concentrations (P > 0.10) 24 h after inoculation. There were no clear patterns of change in monocyte or basophil concentrations after the M. haemolytica challenge.

Total plasma and acute phase protein data are presented in Table 4. Circulating concentrations of total plasma protein, fibrinogen, and haptoglobin were greater (P < 0.01) in heifers fed MGA. There was also an effect of sampling time (P < 0.01) on fibrinogen and haptoglobin with an elevation (P < 0.01) in concentrations of both proteins 24 h after inoculation that remained greater (P < 0.03) than preinoculation concentrations through the final sampling time.

Insulin, Cortisol, and TNF-

Serum insulin, cortisol, and TNF- concentrations are presented in Table 5. There were no differences (P > 0.21) between treatments in circulating insulin or cortisol concentrations, but there was an effect of sampling time on insulin (P < 0.01), with concentrations at every postchallenge sampling point except 120 h after inoculation being greater (P < 0.01) than the prechallenge concentrations. Serum TNF- concentrations were not different (P = 0.13) between treatments, and there was no effect of sampling time on TNF- (P = 1.00).

View this table: [in this window] [in a new window]   Table 5. Serum insulin, cortisol, and tumor necrosis factor- (TNF-) concentrations of heifers fed 0 or 0.5 mg of melengestrol acetate (MGA) daily for 20 d and inoculated on d 14 with Mannheimia haemolytica

Data for plasma urea N, glucose, triglycerides, lacate, and NEFA concentrations are presented in Table 6. Heifers fed MGA had greater concentrations of plasma urea N (P = 0.02) and lower concentrations of plasma glucose (P < 0.01), but there were no differences between treatments for plasma triglycerides (P = 0.49), plasma lactate (P = 0.08), or serum NEFA (P = 0.57). There were effects of sampling time on plasma lactate (P < 0.01), plasma glucose (P = 0.04), and plasma urea N (P < 0.01). Plasma lactate concentrations were lower (P < 0.01) 72 h after inoculation when compared with preinoculation concentrations and remained lower (P < 0.02) throughout the remainder of the sampling period. Compared with preinoculation concentrations, plasma glucose concentrations were lower (P < 0.03) at every post-challenge observational point except 24 and 120 h after inoculation. Plasma urea N concentrations were lower (P < 0.05) 24, 48, and 138 h after inoculation than preinoculation concentrations. A treatment x sampling time interaction (P < 0.02) was observed for NEFA, with concentrations in heifers fed MGA being higher (P < 0.01) 24 h after inoculation and lower (P = 0.02) 138 h after inoculation than controls.

Heifers fed MGA had a greater incidence of lung lesions (14 of 23 vs. 6 of 24; P < 0.02 using Fisher’s exact test; Figure 1) than controls. Severity of lung lesions also tended to be greater in heifers fed MGA (lung lesion score: 3.08% vs. 1.04 ± 1.04%; P = 0.06) than controls.

View larger version (7K): [in this window] [in a new window]   Figure 1. Percentage of heifers with lung lesions 6 d after being inoculated with Mannheimia haemolytica. Heifers were fed 0 or 0.5 mg of melengestrol acetate (MGA) daily for 20 d and were inoculated on d 14 with M. haemolytica. Effect of treatment was calculated using Fisher’s exact test (P < 0.02; n = 23 and 24 for MGA and no MGA, respectively).

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

All clinical scores in this study were made by visual observation. The observation of no change (P = 0.52) in activity level after M. haemolytica challenge is inconsistent with previous studies (Ames et al., 1985; Vestweber et al., 1990; McBride et al., 1992). Heifers fed MGA had greater (P < 0.01) respiratory rate scores than controls, but the difference between treatments appeared to be relatively small. Heifers experienced an increase (P < 0.05) in respiratory rate 12 and 24 h after inoculation that returned to baseline (P = 0.33) by 48 h after inoculation. This observation is consistent with those made by Ames et al. (1985), in which they observed an increased respiratory rate in calves after experimental infection with M. haemolytica. Ames et al. (1985) and Vestweber et al. (1990) also observed an increase in rectal temperature postchallenge that remained greater 72 h after inoculation. In our study, however, rectal temperature had returned to baseline (P = 0.15) 24 h after inoculation. This may indicate a relative low pathogenicity of the bacteria used in our experiment.

The decreases (P < 0.05) in RBC concentration, hemoglobin, and hematocrit postchallenge are likely due to inflammation of the lung tissue. The observations of greater MCV (P < 0.01) and MCH (P = 0.01) in heifers fed MGA than controls, but no effect of sampling time (P = 1.00), may indicate an overall effect of MGA on erythrocyte parameters; however, all observed values for both treatments were within expected ranges for our laboratory (32 to 51 fL for MCV and 11 to 18 pg for MCH). The literature is lacking in information regarding effects of progestins on MCV and MCH specifically, but there is evidence that medroxyprogesterone acetate improves arterial oxygenation and decreases concentrations of CO₂ in the blood in some diseases in humans (Sutton et al., 1975). This could have an effect on the amount of hemoglobin in the RBC and thus their volume.

The observation of greater (P < 0.01) circulating concentrations of eosinophils in cattle fed MGA than controls was not a response to the challenge, because the effect of sampling time was not significant and concentrations of eosinophils were greater in MGA-fed heifers before challenge. Eosinophils are leukocytes that are usually associated with an allergic response or parasitic infection. Progesterone has been shown to increase IL-5 messenger RNA concentrations in T lymphocytes (Wang et al., 1993), and T lymphocytes derived in the presence of progesterone have had an increased ability to produce IL-5 than T lymphocytes that have been derived in the absence of progesterone (Piccinni et al., 1995). Interleukin-5 is a cytokine that induces eosinophil formation and differentiation (Bousquet et al., 1990; Egan et al., 1996). Treatment differences in eosinophil concentrations were small, and the biological significance remains to be determined.

Heifers fed MGA had circulating concentrations of WBC that were outside the expected ranges for our laboratory (4,000 to 12,000/µL) 12 (12,530 ± 952/µL) and 24 (13,030 ± 1,069/µL) h after inoculation. Cattle fed no MGA had elevated (P < 0.02) concentrations of circulating WBC postinoculation, but the increase was of smaller magnitude, and all observed averages were within the expected range for our laboratory. This rise in WBC was due in large part to an increase in circulating segmented neutrophils. Heifers from both treatment groups had concentrations of circulating segmented neutrophils that were outside the expected range (1,000 to 5,000/µL) for our laboratory 12 (MGA = 6,928 ± 1,018/µL; no MGA = 5,294 ± 989/µL) and 24 (MGA = 7,147 ± 1,124/µL; no MGA = 5,101 ± 1,106/µL) h after inoculation, although the heifers fed MGA had overall greater (P < 0.01) concentrations of circulating segmented neutrophils than heifers fed no MGA. These findings are consistent with an earlier study in which heifers fed MGA were shown to have greater concentrations of segmented neutrophils 240 m after injection of Escherichia coli LPS than controls (our unpublished data). Reduced expression of E-selectin on endothelial cells stimulated with IL-1 in the presence of progesterone has been observed (Aziz and Wakefield, 1996). A reduction in the concentration of soluble vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin in blood samples of postmenopausal women receiving estrogen has also been observed following medroxyprogesterone acetate treatment, indicating lower expression of those molecules on the endothelium, because they are shed from endothelial cells within 24 h (Wakatsuki et al., 2002). These reductions may be the indirect result of progesterone and synthetic progestins decreasing inflammation, because it has been reported that progesterone has no effect and some synthetic progestins increase intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression (Tatsumi et al., 2002). It is possible that the increased neutrophilia observed in cattle fed MGA was caused by a decrease in cellular adhesion molecule expression and a subsequent decrease in neutrophil influx into the lung tissue. A lower influx of neutrophils early after challenge may have allowed the bacteria to proliferate more readily in the pulmonary tissue of heifers fed MGA and could explain the increase in incidence of lesions 138 h after inoculation in heifers fed MGA than in the controls. The possible greater severity of the initial inflammatory response in the heifers not fed MGA could have allowed those animals to clear the pathogen early in the postchallenge period, causing resolution of the inflammatory response and subsequent lower incidence of lung lesions 138 h after inoculation.

Apparent contrasting outcomes observed by Slocombe et al. (1985) and Youssef et al. (2004) highlight the complexity of the host-bacterial interactions. In the study conducted by Slocombe et al. (1985), neutrophil-depleted calves were grossly normal 5 h after inoculation with M. haemolytica, whereas lungs of control calves developed lesions, necrosis of the alveolar walls, and other signs of tissue damage after challenge. In contrast, in the experiment conducted by Youssef et al. (2004), the induction of neutrophilia in calves provided some protection against lesions 5 d after inoculation, possibly through increased pathogen clearance.

Mean lung score tended (P = 0.06) to be greater in heifers fed MGA, but when lung lesion scores for only those heifers exhibiting lesions were compared (n = 14, MGA; n = 6, no MGA), they were similar between treatments (MGA = 5.10 vs. no MGA = 4.46). This would suggest that rather than MGA having proinflammatory effects, heifers fed MGA were more susceptible to infection, and thus, a greater incidence of lesions was observed in those heifers, whereas the percentage of involvement of the lungs in heifers afflicted with lesions was similar between treatments.

Increased serum TNF- would be expected after M. haemolytica challenge (Yoo et al., 1995; Morsey et al., 1999; Lafleur et al., 2001), but no postchallenge increases in TNF- were detected. A possible explanation of this includes low virulence of the bacteria used, as evidenced by the low severity of lung lesions. The challenge may have been insufficient to stimulate greater systemic concentrations of TNF-. Another explanation may be a deficiency in the sampling protocol. Horadagoda et al. (1994) found that after M. haemolytica challenge, TNF- concentrations in calves peaked 2 h after inoculation and returned to baseline concentrations 6 h after inoculation. Progestins have been shown to cause a reduction in TNF- production by LPS-stimulated mouse macrophages (Miller and Hunt, 1998). The apparent lack of an effect of MGA on serum TNF- does not rule out the possibility that MGA decreased secretion of TNF- or other proinflammatory cytokines by blood cells in the lung, but this cannot be determined from our study, because TNF- concentrations in the lung were not measured.

There are several possible explanations for the apparent discrepancies between results of this study and those observed by Sulpizio et al. (2003). In the Sulpizio et al. (2003) study, heifers were subjected to natural exposure of BRD, which is often characterized by immunosuppression caused by environmental stressors coupled with a primary viral infection that allows a secondary bacterial infection to establish in the pulmonary tissues. In the current study, methods used sought to model a bacterial infection with no concomitant stressors. The trend for a positive outcome from feeding MGA to prepubertal heifers during the receiving period in the Sulpizio et al. (2003) study may have been the result of effects of MGA on the immune response to a respiratory pathogen other than M. haemolytica or in an alteration of the immunosuppression that often accompanies activation of the hypothalamopituitary-adrenocortical axis.

Results observed by Sulpizio et al. (2003) would have included both the innate and adaptive immune responses to the disease, whereas results from our study only examined aspects of the innate immune response to the bacteria, because there was not sufficient time postinoculation for the calves to mount an adaptive immune response (McBride et al., 1992). There is evidence that the shift from production of T helper type 1 cytokines to T helper type 2 cytokines in pregnant women is caused by progesterone (Piccinni et al., 1995; Szekeres-Bartho and Wegmann, 1996), which may cause alterations in antibody production as well as lymphocyte differentiation.

Differences in the severity of disease challenge may provide further explanation for apparent inconsistencies in results of the 2 experiments. Heifers in the study conducted by Sulpizio et al. (2003) experienced a morbidity rate of 75.6% and a mortality rate of 9.9%, which would indicate a severe natural challenge, because the average incidence of morbidity and mortality are 8 and 1%, respectively (Edwards, 1996). Heifers in the current study experienced an average lung lesion score of 2.1%, indicating a relatively low severity of the challenge. In the case of severe BRD, inflammation that is a result of the pulmonary tissue damage caused by the initial inflammatory response to the pathogen can be substantial. Products produced in the inflammatory response appear to cause more inflammation, as evidenced by the intensification of the response when antiinflammatory cytokines such as IL-10 and IL-13 are blocked by antibodies to either (Ward, 2003). Pulmonary inflammation in severe BRD may be highly exaggerated, far exceeding levels required for pathogen clearance. In such cases, a reduction in the overall inflammatory response would be beneficial to the animal. In the case of the apparent low severity bacterial infection observed in this study, a possible reduction in the inflammatory response caused by MGA could have allowed the bacteria to proliferate more readily in the pulmonary tissues, thus causing more lesions 138 h after inoculation as discussed previously. Observations by Wulster-Radcliffe et al. (2003) that both endogenous and exogenous progesterone increased the susceptibility of gilts to E. coli and Arcanobacterium pyogenes infections support this suggestion.

Melengestrol acetate increased incidence and severity of lung lesions in heifers subjected to a mild experimental challenge with M. haemolytica. Considering results of this study, reasons for the previously observed improvements from feeding MGA during the first 35 d after arrival in the feedlot on growth and the reduction of chronic sickness in heifers with more severe, naturally occurring, undifferentiated respiratory disease remain unclear. The outcome of this study emphasizes the complexity of host-bacterial interaction, and further studies need to be conducted to examine effects of MGA on respiratory disease in cattle.

	Footnotes

 
¹ Contribution number 06-231-J from the Kansas Agricultural Experiment Station.

² Corresponding author: jdrouill{at}oznet.ksu.edu

Received for publication June 21, 2006. Accepted for publication February 22, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


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