Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica

Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica

The Veterinary Journal 192 (2012) 112–119 Contents lists available at ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate...

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The Veterinary Journal 192 (2012) 112–119

Contents lists available at ScienceDirect

The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl

Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica Kuldeep Singh a,⇑, Anthony W. Confer a, Douglas L. Step b, Theresa Rizzi a, John H. Wyckoff III a,1, Hsin-Yi Weng c, Jerry W. Ritchey a a b c

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA Boren Veterinary Teaching Hospital, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA Department of Pathobiology, College of Veterinary Medicine, 2001 South Lincoln, Urbana, IL 61802, USA

a r t i c l e

i n f o

Article history: Accepted 12 May 2011

Keywords: Cytokines Leukotoxin (leucotoxin) Mannheimia haemolytica RT-PCR

a b s t r a c t The objective of this study was to assess the role of leukotoxin (LKT) in modulating the pulmonary cytokine response of calves challenged with Mannheimia haemolytica. Thirty-six calves, seronegative to LKT and M. haemolytica whole cell antigen were divided into three groups (I, II and III). Calves in groups I and II were challenged by the intra-bronchial route with 25 mL of phosphate buffered saline (PBS) containing 0.44  109 cfu/mL of LKT deficient (lkt ) and 25 mL of PBS containing 0.31  109 cfu/mL of wildtype (wt) M. haemolytica serotype 1, respectively. Group III calves were challenged intra-bronchially with 25 mL of sterile PBS. Leukocytes were collected from broncho-alveolar lavage fluid (BALF) 4 days before and at 1, 3, and 6 days post-inoculation (p.i.). Expression of the following cytokines in the recovered leukocytes was measured using real-time PCR: interleukin (IL)-1b, -8, -10, -12 (p40) and TNF-a. The amount of TNF-a produced was also quantified by ELISA. Although a statistically significant difference in the expression of these cytokines was not observed between groups challenged with the wt and lkt strains, the wt infected group (II) did exhibit higher mean clinical scores. Overall, there was considerable variation in the composition of the BALF between the groups and by day 7 p.i., both lkt - and wt-challenged calves had seroconverted to M. haemolytica. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction Mannheimia haemolytica is the major cause of the fibrinous and necrotizing pleuropneumonia of cattle termed bovine pneumonic pasteurellosis (BPP) or ‘shipping fever’. Several virulence factors of M. haemolytica promote host-pathogen interactions by enabling bacterial colonization of the lungs and contribute to the development of pneumonia (Highlander, 2001). Leukotoxin (LKT) and lipopolysaccharide (LPS) are key factors in this context (Confer et al., 1990). Most of the knowledge of the role of LKT and LPS in the pathogenesis of BPP has come from in vivo studies using purified LKT alone or in combination with LPS (Stevens and Czuprynski, 1995; Marcatili et al., 2002). Since the effects of LKT and LPS are synergistic, their roles are better appreciated when used in combination (Lafleur et al., 2001). In vivo studies using whole bacteria are

⇑ Corresponding author. Present address: Department of Pathobiology, College of Veterinary Medicine, 2001 South Lincoln, Urbana, IL 61802, USA. Tel.: +1 217 2440133. E-mail address: [email protected] (K. Singh). 1 Present address: Merial Ltd., Research and Development, 115 Trans Tech Drive, Athens, GA 30601-1649, USA. 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.05.015

limited, and, to date, only two studies using LKT deletion mutant organisms (lkt ) have been conducted (Tatum et al., 1998; Highlander et al., 2000). These studies found reduced clinical and lung lesion scores in animals infected with lkt mutants compared to those challenged with wild-type (wt) strains of M. haemolytica. Lower lesion scores were characterized by reduced necrosis and lower numbers of degenerate neutrophils (Tatum et al., 1998; Highlander et al., 2000). However, Highlander et al. (2000) could only demonstrate a partial reduction in the virulence of the lkt mutant. The molecular basis of this attenuation remains to be elucidated. Given the key role of inflammatory cytokines in the pathogenesis of BPP (Lafleur et al., 2001; Malazdrewich et al., 2001), a profile of the cytokines expressed in response to infection would likely enhance our understanding of the important events at play. In this study we chose to quantify tumor necrosis factor (TNF)-a and interleukin (IL)-1b and -8 as these are pleiotropic, early response pro-inflammatory mediators that are produced by a variety of cells (Malazdrewich et al., 2001). IL-10 and -12 were selected because of their role in regulating the adaptive immune response. Our objective was to investigate the role of LKT in modulating cytokine gene and protein expression in bovine leukocytes obtained from the lungs of calves experimentally infected with isogenic LKT-deficient (lkt ) and with wt strains of M. haemolytica

K. Singh et al. / The Veterinary Journal 192 (2012) 112–119

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serotype 1, respectively. The wt strain was isolated from a pneumonic bovine lung and was found to be more virulent than existing strains cultured from cattle in the Oklahoma region of the USA (unpublished data). The lkt strain was prepared by allelic replacement of the entire lktA and part of the lktC genes with the beta-lactamase enzyme gene (Murphy et al., 1995). A ‘mixed regression’ model was used in the hypothesis testing.

On Western blotting, a monoclonal antibody MM 605 (kindly provided by Dr. S. Srikumaran, Washington State University) recognized the 102 kDa LKT band and associated breakdown bands in the supernatant obtained from the culture of wt M. haemolytica, whereas a similar band was not detected in the culture supernatant obtained from lkt strain (Singh et al., 2011). The growth of the wt strain on 5% sheep blood agar was accompanied by a distinct zone of hemolysis surrounding the colonies. No such zone was associated with growth of the lkt -strain (data not shown). Bovine alveolar macrophages (BAM) challenged with wt strain exhibited 43% more cytotoxicity compared to cells challenged with the lkt strain (Singh et al., 2011).

Materials and methods

Experimental protocol

Preparation of inoculum

The study was conducted under the guidelines of Oklahoma State University Institutional Animal Care and Use Committee (protocol number VM50182). Thirty-six, 140–200 kg, male and female, cross-breed calves were screened for antibodies against LKT and M. haemolytica whole cell (WC) antigens at 18 days prior to inoculation using an ELISA (Confer et al., 1985). Healthy calves with minimal antibody concentrations were selected, transported to the study site 10 days prior to the commencement of the experiment, and were allowed to acclimatize. Calves were randomly assigned to three groups (I, II and III) of 12 animals/group. Group I and II calves were challenged intra-bronchially with lkt and wt M. haemolytica strains, respectively. Group III animals served as controls and were challenged with sterile PBS. Calves from the different groups were held in individual pens located so as to avoid the aerosol transfer of bacteria. On day 0, calves in groups I and II received 25 mL of suspension in PBS containing 0.44  109 cfu/mL of the lkt and 0.31  109 cfu/mL of the wt strains, respectively. These were delivered intra-bronchially using an endotracheal tube followed by a 25 mL PBS ‘flush’. Bronchoalveolar lavage fluid (BALF) was obtained from each calf 4 days before infection and at 1, 3 and 6 days post-inoculation (p.i.) using 180 mL of sterile PBS via an endotracheal tube with the instilled fluid retrieved by suction. On average, less than 50% of the instilled fluid was collected in 50 mL tubes containing 1% penicillin–streptomycin and amphotericin B (Cambrex) and was transported to the laboratory on ice. The tubes were then centrifuged at 500 g for 7 min and 500 lL of supernatant was collected for ELISA. The cell pellet was gently washed in 10 mL of PBS and then centrifuged again in 15 mL of PBS at 500 g for 7 min. RNA was extracted from the cell pellet as previously described (Rottman et al., 1996). From 1 day prior to inoculation to 6 days p.i., the calves were clinically evaluated daily and given a clinical ‘score’ based on a nonparametric scale (Tatum et al., 1998). Scores of between ‘0’ and ‘4’ were allocated based on the following criteria: rectal temperature >39 °C; evidence of depression; and dyspnea or respiratory rate P60/min.

Both the wt and lkt strains were cultured in a similar fashion and had almost identical growth curves. Briefly, bacteria were grown overnight on brain heart infusion (BHI) agar containing 5% sheep blood (Hardy Diagnostics), both without and with added ampicillin (10 lg/mL) for the wt and lkt cultures, respectively. Multiple colonies were subsequently transferred into 150 mL of BHI broth and incubated for 6 h at 37 °C at 70 oscillations/min (opm) in a shaking incubator. The broth culture was centrifuged twice at 8000 g for 15 min. The bacterial pellet was washed with 10 mL of phosphate buffered saline (PBS). After a second centrifugation, the pellet was re-suspended in RPMI 1640 media or PBS and the concentration quantified by spectrophotometry (Ultraspec 2000, Pharmacia). In our previous experiments, an optical density (OD) of 0.72–0.74 at 650 nm corresponded to a concentration of approximately 109 colony forming units (cfu)/mL (Confer et al., 2006). The exact concentration of the viable bacterial suspension was subsequently confirmed by standard colony count at various dilutions on BHI blood agar as 0.44  109 cfu/mL and 0.31  109 cfu/mL for the lkt and wt strains, respectively.

Leukotoxin production Both wt and lkt strains were grown on BHI supplemented with 5% sheep blood agar. A single colony was suspended in BHI broth and incubated overnight at 37 °C in a shaking incubator at 160 opm. The culture was subsequently re-suspended in 1 L of RPMI 1640 (R7509 Sigma) and incubated at 37 °C at 160 opm. The culture was centrifuged at 3800 g for 20 min and the supernatant was filtered using 0.2 lm filters (Fisher 167-0020). The concentrate was then repeatedly centrifuged (Amicon ultra-15, Millipore) at 2000 g for 15 min. The culture supernatant containing semi-purified LKT was aliquoted and stored at 86 °C.

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Days p.i. Fig. 1. Mean (±SE) clinical score (a) and body temperature (b) of calves challenged with wildtype (wt) and leukotoxin deficient (lkt ) strains of Mannheimia haemolytica and of uninfected negative control animals from prior to challenge until 7 days post-inoculation (p.i.).

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Broncho-alveolar lavage procedure Broncho-alveolar lavage (BAL) was a slight modification of the technique developed by Corstvet et al. (1982). The calves were restrained in a chute with their heads extended and elevated to minimize the angle between the nares and larynx. A 244 cm long silicone BAL catheter (Equine Bronchoalveolar Lavage Catheter, Bivona Medical Technologies), of 11 mm outer and 3 mm inner diameter, was passed through the nares, nasopharynx and larynx, and into the trachea to the level of the tracheal bifurcation. At this point the catheter was rotated approximately one-quarter to one-half turn to facilitate its advancement into one of the main stem bronchi. The catheter was advanced into a distal airway, approximately 238 cm from the external nares. The cuff was inflated with 6–10 mL of air to seal the airway. The lung was lavaged by delivery and immediate recovery of three, 60 mL instillations of PBS containing 1% calf serum. The recovered suspension was placed in a sterile centrifuge tube and delivered to the laboratory on ice. An aliquot of the suspension was removed to perform total and differential cell counts using cytospin followed by Wright’s staining. The suspension was centrifuged and the cell pellet washed twice with PBS, The total cellular RNA was immediately extracted.

Serological response to Mannheimia haemolytica antigens Serum IgG to M. haemolytica serotype 1 LKT and WC antigens were determined by ELISA as previously described with minor modification (Confer et al., 1985). Briefly, LKT was prepared as described above. To obtain WC antigen, formalin-killed M. haemolytica was prepared from a washed, 24 h culture by suspending cells in 0.4% formalinized saline at a concentration determined spectrophotometrically to be 1.850 at OD650 (Confer et al., 1997). Wells of 96 well polystyrene EIA/RIA microtiter flat-bottom, high-binding plates (COSTAR 9018 Corning Inc.) were coated with LKT at 50 ng/well at a concentration equivalent to 109 CFU. Primary antisera were diluted in blocking buffer solution consisting of 1% bovine serum albumin and 0.05% Tween-20 in PBS (pH 7.4) and were assayed in triplicate. Bound antibody was detected using peroxidase-labeled goat monoclonal antibody to bovine IgG (H + L). Enzymatic activity was assayed using the color substrate ortho-phenylene

Antibody response (ng)

(a)

diamine (Amresco, 5348-50T). The ODs of the wells were read at 650 nM. Antibody responses were expressed as ng of IgG binding compared to a standard immunoglobulin curve.

Real-time RT-PCR and ELISA The mRNA of multiple cytokines was determined by real time RT-PCR. Briefly, total RNA was extracted as described previously (Rottman et al., 1996) and cDNA was generated using a reverse transcriptase kit (QuantiTect Rev. Transcription Kit, Qiagen). RNAstat-60 (Tel-Test B) was added to the pelleted cells and the cells were then lysed by repeatedly pipetting the suspension up and down. The RNA was subsequently isolated according to the manufacturer’s instructions, was measured using a spectrophotometer, and adjusted to 0.1 lg/lL with diethyl pyrocarbonate (DEPC)-treated water. Real-time RT-PCRs were performed in individual 200 lL Eppendorf tubes containing 12.5 lL of 1 TaqMan Universal PCR master mix, 1.5 lM of each primer, 10 lL of water, and 2.0 lL of sample cDNA to a final volume of 26 lL. PCR amplification and detection were performed on an ABI Prism 7000 Sequence Detection System using the following cycling conditions: 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. All RT-PCRs were carried out in duplicate with appropriate controls on each plate. Bovine b-actin mRNA was used as endogenous reference mRNA. The sequences of the primers used were previously published (Caswell et al., 1998). The geometric mean fold increase in cytokine expression was calculated as described previously (Wyckoff et al., 2005). Briefly, the formula 2T DDC was used to calculate the relative amount of target gene expression level, where DDCT = DCT,q DCT,cb. The term DCT,q represents the difference in threshold cycle number of the target and reference (b-actin) genes using cDNA from BAM challenged with either the lkt or wt strains and DCT,cb is the difference in threshold cycle number of the target and reference genes using cDNA from untreated BAM. Thus, data for each cytokine were normalized to that obtained for b-actin in each respective treatment and were presented as fold increases in mRNA levels due to treatment relative to cells from control animals. A TNF-a ELISA was performed according to the manufacturer’s instructions (Bovine TNF-alpha screening, Endogen).

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Days p.i. Fig. 2. Serum immunoglobulin (Ig) G response to M. haemolytica leukotoxin (LKT) (a) and whole cell (WC) lysate (b) as measured by ELISA from prior to challenge until 7 days post-inoculation (p.i.).

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Results Clinical scoring and serology The lkt - and wt-challenged calves had a higher mean clinical score relative to controls throughout the study (P = 0.060). However, the average of these scores was not significantly different (P = 0.423) in the calves challenged with either the lkt or wt strains (Fig. 1a). From day 3 to 7 p.i., wt-challenged calves exhibited slightly higher mean clinical scores compared to

(a)

lkt -challenged calves (Fig. 1a). In contrast to other calves in the wt-challenged group, one animal (number 21), developed severe dyspnea, coughing, depression and pyrexia and died within 72 h p.i. At necropsy, gross and microscopic lesions typical of the fibrinonecrotic bronchopneumonia induced by M. haemolytica infection were observed. Large numbers of M. haemolytica and small numbers of Pasteurella multocida organisms were cultured from the affected lungs. Both lkt - and wt-challenged calves had slightly higher mean (P = 0.121) rectal temperatures from days 1 to 5 p.i. relative to controls (Fig. 1b).

Compsition of BAL fluid from control

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Days p.i. Fig. 3. Composition of broncho-alveolar lavage (BAL) fluid obtained from: (a) group III calves (controls) given sterile phosphate buffered saline (PBS) intra-bronchially; (b) group I calves given leukotoxin deficient Mannheimia haemolytica intra-bronchially; and (c) group II calves given wild-type M. haemolytica intra-bronchially, from prior to challenge until 6 days post-inoculation (p.i.).

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All calves in the three experimental groups had low antibody responses to both LKT and WC antigen 18 and 4 days prior to inoculation. By day 7 p.i. all calves in groups I and II had seroconverted to both these antigens (Fig. 2a and b). Analysis of bronchoalveolar lavage fluid At day 4 prior to challenge, cells in the BALF consisted predominantly of alveolar macrophages (75–89%) admixed with smaller numbers of neutrophils (5.7–25%), lymphocytes (1.5–44.9%) and, rarely, eosinophils (Fig. 3a–c). On day 1 p.i., BALF from controls and from wt-challenged calves contained predominantly neutrophils (61.9% and 55.24%, respectively), admixed with macrophages (42.4% and 36.8%, respectively), a small population of lymphocytes (1.3% and 2.1%, respectively), and rare eosinophils (Fig. 3a and b). In contrast, BALF from lkt -challenged calves at this time-point primarily contained alveolar macrophages (96%) admixed with rare lymphocytes, neutrophils, and eosinophils (Fig. 3c). By day 3 p.i., the composition of the BALF from the lkt - and wt-challenged calves was 50.9% and 45.8% macrophages, 44.8% and 52.2% neutrophils, and 4.1% and 1.9% lymphocytes, respectively (Fig. 3b and c). By day 6 p.i. leukocyte proportions in BALF were similar to prechallenge: 53.7–67.4% alveolar macrophages; 28.7–38.5% neutrophils, 3.0 10.6% lymphocytes and 0.1–0.8% eosinophils (Fig. 3a–c). Quantification of cytokine expression in broncho-alveolar lavage fluid leukocytes Real-time PCR was used to assess mRNA expression of the following cytokines in the leukocytes obtained from BALF of the calves challenged with lkt and wt strains of M. haemolytica: TNF-a, IL-1, 8, -10, and -12 (p40). Increased expression of mRNA of the proinflammatory cytokines IL-1, -8, and TNF-a was observed on day 1 p.i. compared to 4 days prior to inoculation and relative to expression levels at days 3 and 6 p.i. However, the expression of IL-1 was increased on day 3 p.i. in leukocytes from wt-challenged calves (Fig. 5a–c). Increased baseline expression of IL-1, compared to days 1 and 6 p.i., was observed 4 days prior to inoculation (Fig. 5b), and a mild increase was noted on day 3 p.i. in wt-challenged animals. No statistically significant differences were found in the expression of IL-1, IL-8, and TNF-a, between lkt - and wt-challenged calves (P = 0.224, 0.630, and 0.239, respectively). There was 3.6-fold increased expression of IL-10 mRNA on day 1 p.i. in leukocytes from group I calves compared to baseline values 4 days prior to challenge. No significant difference (P = 0.064) was

Mean TNF-α protein concentration (pg/mL)

lkt-strain of Mannheimia haemolytica wt strain of Mannheimia haemolytica Negative control

Days p.i. Fig. 4. Quantification by ELISA of tumor necrosis factor (TNF)-a protein (pg/mL) in bronchoalveolar lavage fluid from calves challenged with leukotoxin deficient (lkt ) and wild type (wt) strains of Mannheimia haemolytica and from negative controls from prior to challenge until 6 days post-inoculation (p.i.). Data represent mean (±SEM) from 12 calves/group.

observed between lkt - and wt-challenged calves (Fig. 5d). There was unexpected increased expression of IL-12 mRNA 4 days prior to challenge in calves subsequently inoculated with the lkt strain. This value had declined by days 1 and 3 p.i. with a subsequent slight increase at day 6 p.i. For wt-challenged calves, increased IL-12 expression had decreased by days 1 and 6 p.i. although there was of a mild increase on day 3 p.i. (Fig. 5e). No statistically significant differences (P = 0.718) were observed between lkt and wtchallenged animals. Quantification of TNF-a in broncho-alveolar lavage fluid by ELISA Four days prior to challenge, 70–80 pg/mL of TNF-a protein was present in the BALF of all calves. This increased to 90–95 pg/mL in all groups at day 1 p.i., but by day 3 p.i. had decreased to 65–69 pg/mL and remained at this level until day 6 p.i., except for wt-challenged calves where a further reduction to 54 pg/mL was observed (Fig. 4). No statistically significant differences were found between lkt - and wt-challenged calves (P = 0.333). Discussion Numerous in vitro studies have highlighted the virulence of LKT and lipopolysaccharide (LPS) (Sibille and Reynolds, 1990; Maheswaran et al., 1992; Lafleur et al., 2001), although studies assessing whole bacteria containing the full repertoire of virulence factors are limited. Following intra-bronchial challenge of calves with lkt and wt strains of M. haemolytica, we were surprised to find no statistically significant difference in the expression of various cytokines by leukocytes obtained by BAL from the two infected groups of animals. This was unexpected given the putative critical role of LKT in the pathogenesis of pneumonia caused by M. haemolytica. In an experimental model of bovine pneumonic pasteurellosis Highlander et al. (2000) and Tatum et al. (1998) demonstrated a reduction in the clinical and lung lesion scores by using lkt mutants of M. haemolytica. Tatum et al. (1998) used an isogenic LKTdeficient M. haemolytica which produced mild clinical signs and had a decreased virulence in calves. The lungs of calves challenged with wt M. haemolytica strains contained numerous degenerate and ‘streaming’ intra-alveolar neutrophils (so-called ‘oat’ cells) accompanied by fibrin and parenchymal necrosis. In contrast, the lungs of calves challenged with a lkt A mutant did not exhibit such neutrophil degeneration (Tatum et al., 1998). Subsequently, reduced grossly visible pulmonary and pleural pathology was observed in calves challenged by the intra-thoracic route with a lkt C mutant compared to those inoculated with a wt strain. Unfortunately, analysis of BALF and quantitative cytokine evaluation was not performed in these studies. The fact that a statistically significant difference was not found in our study may explain the lack of difference in pulmonary pathology in calves challenged with LKT-deficient M. haemolytica previously observed (Highlander et al., 2000). Alternatively, the lack of consensus on how attenuated LKT-deficient strains of M. haemolytica actually are in vivo could reflect the role of other virulence factors such as LPS, which is known to induce the production of similar cytokines to those triggered by LKT. It is thus possible that any potential difference in cytokine production induced by lkt and wt strains in this study was overwhelmed by the effect of LPS (Lafleur et al., 2001). Our findings suggest that although LKT contributes to the pathogenesis of shipping fever, it is possible that other virulence factors also participate. This hypothesis is supported by studies on the role of another RTX exotoxin, E. coli hemolysin (Welch et al., 1992; Moxley et al., 1998). Work by Highlander et al. (2000) and Tatum et al. (1998) indicate that while mutation of lkt A created a highly attenuated

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Days p.i. Fig. 5. Graphs quantifying interleukin (IL)-8 (a), IL-1 (b), TNF-a (c), IL-10 (d), and IL-12 (p40) (e) mRNA expression by pulmonary leukocytes obtained by broncho-alveolar lavage of calves challenged with leukotoxin deficient (lkt ) and wild type (wt) strains of Mannheimia haemolytica, and from negative controls, from prior to challenge until 6 days post-inoculation (p.i.). Expression data were normalized for the expression of b-actin and increases are expressed as fold increases. The data represent the geometric means (±SE) of 12 animals/group.

M. haemolytica strain, mutation of lkt C resulted in only a subtle reduction in virulence. The LKT mutant used by Highlander et al. (2000) was created by inserting bacteriophage P1 loxP within the lkt C open reading frame, an insertion that resulted in a ‘frameshift’ mutation but did not alter the expression of other LKT genes such as lktA, lktB, and lktD. The LKT mutant created by Tatum et al. (1998) was constructed by allelic replacement of lktA. The lktC gene encodes a transacylase that post-translationally modifies the

inactive pro-LKT A to biologically active LKT whereas the lktA gene encodes the inactive pro-LKT A. Further characterization suggested the LKT mutant, created by lktA and lktC mutations, is not capable of activating neutrophils, inducing IL-8 expression, or of causing cytolysis (Thumbikat et al., 2003). Given that the lkt strain used in the present study was generated by targeting both lktA and lktC, it is highly unlikely that LKT secreted by the lkt strain was responsible for inducing cytokine production by leukocytes.

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In a recent in vitro experiment we demonstrated that BAM challenged with a similar lkt strain to that used in the current study, resulted in increased quantities of TNF-a and IL-10 proteins in the supernatant compared to wt challenged cells (Singh et al., 2011). Similarly, using real-time RT-PCR, increased mRNA expression of TNF-a, IL-1b, -8, -10 and -12 was found in lkt -challenged compared to wt-challenged BAM. This difference in cytokine production was attributed to cell cytotoxicity induced by the wt-strain. Although LKT-induced necrosis was observed histologically in the lungs of a wt-challenged calf, it is possible that LPS stimulation of inflammatory cells had an over-riding influence on the reduced cytokine expression due to LKT-induced leukocyte cytotoxicity. It is also possible that the results obtained in the current study do not reflect the true concentration of pulmonary cytokines. The corroboration of cytokine expression using other techniques such as in situ hybridization on lung sections and using ELISA would be helpful in this context. The migration and activation of neutrophils within pneumonic lungs are regulated by a complex network of interactions between cytokines, leukocytes, vascular endothelium, cellular adhesion molecules, and soluble chemotactic factors (Malazdrewich et al., 2001). Current evidence indicates that pro-inflammatory cytokines, in particular IL-8, are central to the initiation and orchestration of neutrophil migration (Caswell et al., 1998; Mitchell et al., 2003). In vitro studies indicate that biologically active LKT induces increased expression of IL-8 in BAM (Thumbikat et al., 2003). In the current study, BALF obtained from lkt -challenged calves on day 1 p.i. contained <2% neutrophils in contrast to the neutrophil-rich content of the BALF recovered from wt-challenged animals. This difference may be due to the inability of the lkt -strain to produce biologically active LKT. The decreased numbers of neutrophils found support the finding of reduced pulmonary injury in previous in vivo studies using LKT mutants (Tatum et al., 1998; Highlander et al., 2000). In line with these observations we observed an increased mean clinical score and death of one of the wt-challenged calves likely linked to increased IL-8 production and attendant neutrophil infiltration. Slocombe et al. (1985) demonstrated that neutrophil-depleted calves were little affected by M. haemolytica infection compared to calves with a normal complement of these granulocytes. IL-10 is a multifunctional cytokine central to the termination of the inflammatory response, inhibiting the activation and effector function of T cells and macrophages and regulating the growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, granulocytes, and endothelial cells (Moore et al., 2001). The cytokine suppresses the antigen-presenting functions of macrophages by down-regulating MHC II and co-stimulatory molecules, and by inhibiting IL-12 production. This, in turn, inhibits the generation and/or maintenance of antigen-specific Th1 cells (Buza et al., 2004; Nylen and Sacks, 2007). In the current study, up-regulation of IL-10 by BAL leukocytes from lkt -challenged calves by day 1 p.i. may have masked the subsequent expression of other cytokines by these animals. Similarly, increased expression of IL-10 in lkt -challenged BAM and the subsequent reduction in the concentration of pulmonary cytokines in calves challenged with lkt -compared to wt strains may explain the differences in lung pathology and reduced virulence previously reported (Tatum et al., 1998; Highlander et al., 2000). It is also possible that the lkt -challenged BAM survive long enough to reduce inflammation through IL-10 mediation. Alternatively, the lack of statistically significant differences in cytokine mRNA expression in calves challenged with lkt - or wt-strains could be due to the infecting dose of bacteria used. In the present study this dose was chosen in order to demonstrate altered cytokine production by recovered leukocytes yet not prove lethal to the inoculated calves (Lo et al., 2006). Despite the fact that no statistically significant differences in cytokine production were

observed in the present study, the wt-challenged calves did exhibit higher clinical scores compared to the lkt -challenged animals and one calf inoculated with the wt-strain died with a necrotizing bronchopneumonia typical of M. haemolytica infection after 72 h. The LKT ELISA indicated that the calves were seronegative prior to challenge and only mild seroconversion to LKT was noted in lkt -challenged calves following infection. This suggests that little or no LKT protein was produced by lkt -strain used in this study. The slightly elevated titer to LKT observed in lkt -challenged calves was considered a response to the resident commensal population of M. haemolytica in the nasopharyngeal region of the calves. Mannheimia haemolytica is an opportunistic pathogen and shipping fever is a multifactorial disease in which stress and viral infections play important predisposing roles. In the absence of such predisposing factors, it is difficult to reproduce BPP under experimental conditions (Highlander et al., 2000). Moreover, though intra-bronchial administration of M. haemolytica has been widely used to reproduce BPP experimentally, this approach does not closely mimic natural infection where prior nasopharyngeal colonization is essential. Conclusions Cytokine expression in the BALF leukocytes from calves challenged with LKT-deficient and wt strains of M. haemolytica S1 was measured. Although statistically significant differences in expression were not observed, calves inoculated with the wt-strain had higher mean clinical scores. Our results suggest that bacterial virulence factors such as LPS may, in addition to LTK, contribute to the pathogenesis of BPP through the selective induction of cytokine synthesis by pulmonary leukocytes. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements This research project was supported in part by a grant from the Noble Foundation, Ardmore, Oklahoma, USA. The laboratory support provided by Amy Cragun, Marie Montelongo, Joshua Wray, Jason Thorn, Clayton Smith, and Jessie Holyoak is greatly appreciated. References Buza, J.J., Hikono, H., Mori, Y., Nagata, R., Hirayama, S., Aodon-geril, Bari, A.M., Shu, Y., Tsuji, N.M., Momotani, E., 2004. Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis. Infection and Immunity 72, 2425–2458. Caswell, J.L., Middleton, D.M., Sorden, S.D., Gordon, J.R., 1998. Expression of the neutrophil chemoattractant interleukin-8 in the lesions of bovine pneumonic pasteurellosis. Veterinary Pathology 35, 124–131. Confer, A.W., Panciera, R.J., Fulton, R.W., Gentry, M.J., Rummage, J.A., 1985. Effect of vaccination with live or killed Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis. American Journal of Veterinary Research 46, 342–347. Confer, A.W., Panciera, R.J., Clinkenbeard, K.D., Mosier, D.A., 1990. Molecular aspects of virulence of Pasteurella haemolytica. Canadian Journal of Veterinary Research 54, S48–S52. Confer, A.W., Clinkenbeard, K.D., Gatewood, D.M., Driskel, B.A., Montelongo, M., 1997. Serum antibody responses of cattle vaccinated with partially purified native Pasteurella haemolytica leukotoxin. Vaccine 15, 1423–1429. Confer, A.W., Ayalew, S., Panciera, R.J., Montelongo, M., Wray, J.H., 2006. Recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE enhances commercial M. haemolytica vaccine-induced resistance against serotype 6 challenge. Vaccine 24, 2248–2255.

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