Veterinary Immunology and Immunopathology 161 (2014) 151–160

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Research paper

Neutrophil gelatinase-associated lipocalin (NGAL) and insulin-like growth factor (IGF)-1 association with a Mannheimia haemolytica infection in sheep Yaser H. Tarazi a,∗ , Mohammad S. Khalifeh a , Mohammad M. Abu Al-Kebash a , Mohammad H. Gharaibeh b a Department of Basic Veterinary Medical Science, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan b Department of Animal Protection and Prevention, Faculty of Agriculture, P.O. Box 301, Irbid 26150, Jordan

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Article history: Received 8 March 2014 Received in revised form 31 May 2014 Accepted 28 July 2014 Keywords: Mannheimia haemolytica NGAL IGF-1 TNF-␣ IL-10 Sheep

a b s t r a c t This study was aimed at mapping the tissue distribution of some inflammatory parameters associated with a Mannheimia haemolytica (M. haemolytica) infection in sheep. The M. haemolytica was isolated and characterized from the affected lungs of slaughtered animals. Cytokines such as tumor necrosis factor (TNF)-␣, interleukin (IL)-10, insulin-like growth factor (IGF)-1, as well as the acute-phase protein, neutrophil gelatinase-associated lipocalin (NGAL), were identified in the lung tissues, the serum, and the lymph nodes of M. haemolytica infected sheep, by enzyme-linked immunosorbent assay (ELISA). NGAL and IGF-1 pointed to an innate immune response, and epithelial cell repairing, respectively. The adaptive immune response was identified through the type of cytokines present in the affected sheep, as TNF-␣ represents the pro-inflammatory cytokines, and IL-10 represents the anti-inflammatory cytokines. M. haemolytica isolates were confirmed by polymerase chain reaction (PCR) and DNA sequences. There was a significant difference in the concentrations of NGAL, IGF-1, TNF-␣, and IL-10, as observed in the affected sheep when compared to the healthy sheep. This study, for the first time, closely describes the distribution of some key and new inflammatory parameters in the tissue homogenate of affected lungs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Mannheimia haemolytica is one of the most common respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in calves, sheep, goats, neonatal-weaned and growing lambs, as well as in adult animals (Ackermann and Brogden, 2000; Al-tarazi,

∗ Corresponding author. Tel.: +962 2 7201000x22009/22002; fax: +962 2 7201081. E-mail addresses: [email protected] (Y.H. Tarazi), [email protected] (M.S. Khalifeh). http://dx.doi.org/10.1016/j.vetimm.2014.07.010 0165-2427/© 2014 Elsevier B.V. All rights reserved.

1995). Pneumonia is developed when antibacterial defense mechanisms of the lung break-down and bacterial proliferation occurs (Bruere et al., 2002). The neutrophil gelatinase-associated lipocalin (NGAL) protein, also known in the literature as lipocalin 2, 24p3, and uterocalin, is expressed by many cells and tissues, including the lungs (Daure et al., 2013). NGAL is considered to be a pivotal component of the innate immune response to bacterial infection such as Escherichia coli, Klebsiella, Salmonella, and Mycobacterium tuberculosis (Chan et al., 2009; Martineau et al., 2007; Nairz et al., 2008; Wu et al., 2010). NGAL has the ability to arrest bacterial growth by sequestrating iron-laden siderophores (Flo et al., 2004).

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Insulin-like growth factor (IGF)-1 is a profibrogenic mediator, acting as a potent mitogen, and it also stimulates collagen synthesis by fibroblasts (Allen et al., 1998). It is mainly secreted by alveolar macrophages in the affected lungs (Rom et al., 1988), but it can also be expressed by other cell types, such as fibroblasts and epithelial or endothelial cells (Homma et al., 1995). An increased IGF-1 expression and release has been demonstrated in patients with idiopathic pulmonary fibrosis (Aston et al., 1995), systemic sclerosis (Harrison et al., 1994), coal-worker pneumoconiosis (Vanhee et al., 1995), and pulmonary sarcoidosis in adults (Allen et al., 2008), as well as in interstitial lung disease in children (Chadelat et al., 1998). One of the hallmark histopathological features of an M. haemolytica infection is an extensive infiltration of the lungs with neutrophils (Aulik et al., 2010). A depletion of neutrophils protects animals from subsequent lung injury, suggesting that this cell type is responsible for much of the pulmonary pathology associated with the disease (Wagner and Roth, 2000). The recruitment and activation of neutrophils are regulated by a complex network of interactions between cytokines, leukocytes, vascular endothelium cells, cellular adhesion molecules, and soluble activating and chemotactic factors (Kisiela and Czuprynski, 2009; Leite et al., 2002, 2004). The inflammatory cytokines TNF-␣, IL-1, and IL-8 play a central role in the recruitment and activation of neutrophils (Singh et al., 2012, #24; Redondo et al., 2011, #3). TNF-␣ and IL1␤ are pleiotropic cytokines that are secreted by immune cells, such as monocytes and macrophages, and in a response to a microbial pathogens infection (Malazdrewich et al., 2001). M. haemolytica, and its major virulence factors, are potent inducers of TNF-␣ and IL-1␤ proteins in alveolar macrophages (Aulik et al., 2010; Lafleur et al., 2001; Singh et al., 2011). On the other hand, IL-10 is a regulatory anti-inflammatory cytokine that is suspected to play a role in resolving certain stages of pneumonia. In the present study, the presence and characterization of M. haemolytica in the pneumonic lungs of sheep were investigated; the immune response in affected sheep was also assessed. Inflammatory parameter distribution, in pneumonic and healthy lung portions, regional lymph nodes, and in serum, was outlined in affected sheep, as well as in those samples that were collected and compared with the samples collected from healthy animals. 2. Materials and methods 2.1. Isolation and identification of M. haemolytica One hundred and fifty pneumonic lungs specimens were collected from 6 to 12 months old Awassi sheep of both sexes. Tissue specimens were taken from the consolidated lung, margins of gross pneumonic lesions of 150 sheep and 7 healthy lungs were used as control for bacteriological examination. In addition, specimens, from the bronchial or mediastinal lymph nodes, and the serum, of the affected and healthy animals, were collected. All samples were collected from Amman and Irbid municipality slaughter houses. As a positive control, M. haemolytica

isolates were compared with reference bacterial strains (NCTC #10627). Lung tissue specimens, and the reference strain, were cultured on a columbia blood agar base (Oxoid, U.K.) containing 5% sheep blood, and on a MacConkey agar base (Oxoid, U.K.). The inoculation and incubation were done as described previously by Quinn et al. (1994). Gram’s staining of M. haemolytica shows a Gram negative rod, or coccobacilli, and occasionally a pleomorphic cell with a bipolar characteristic. For the confirmation of identification, an API 20 NE (REMEL RAPID System, USA) was used according to the manufacturer’s instructions and the results were interpreted using a software program. The identified bacteria were stored at −70 ◦ C for further use. The conventional identification of M. haemolytica isolates were also confirmed by PCR using PHSSA primers (F: 5 -TTC ACA TCT TCA TCC TC-3 and R: 5 -TTT TCA TCC TCT TCG TC-3 ) (Ozbey et al., 2004). DNA was extracted using a Promega Wizard® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer’s instructions. The PCR reaction was performed in a total volume of 25 ␮L, containing 1X Go Taq reaction buffer, 2.5 mM MgCl2 , 0.4 ␮M from each primer, 0.2 mM dNTPs (Bio Basic Inc., Canada), 1.25 U from Go Taq DNA polymerase (Promega Corporation, Madison, WI, USA), and 1 ␮L from the extracted DNA. The DNA was amplified under the following conditions in a PTC200 type thermocycler (MJ Research Inc., USA): denaturation at 94 ◦ C for 2 min, 40 cycles using the following settings: denaturation at 94 ◦ C for 45 s, annealing at 45 ◦ C for 45 s and extension at 72 ◦ C for 1 min, followed by 5 min at 72 ◦ C. The PCR product was analyzed on a 1.5% agarose gel stained with 1 ␮g/ml ethiduim bromide (Promega, USA). Two DNA ladders, 1 kb and 100 bp (Bio Basic Inc., Canada), were used to determine the size of the amplified fragments. One of each primer’s prepared PCR products, as obtained in this study, were sent to (Sequetech, California, USA), to determine their base sequences, by the utilization of their Biosystems 3730xl DNA Analyzer. The obtained results were then compared with the available sequences in GenBank using an NCBI-Blast algorithm. 2.2. Immunity parameters Out of 150 lung tissue samples that were examined, fifty-eight samples (38.7%) had showed M. haemolytica presence. Seven tissue samples, that had typical lesions of the disease, were analyzed for an inflammatory parameter expression. Serum and lymph nodes, corresponding to these selected tissues, were also analyzed. In addition, similar samples from the healthy sheep were collected and compared for an inflammatory parameter expression with M. haemolytica infected tissues. 2.2.1. Tissue homogenates One to two cubic centimeters of a sheep tissue specimen (lung or lymph node), from which M. haemolytica was isolated in pure culture, was placed in a round bottom tube containing 3–5 ml of lysing buffer, fortified with a protease inhibitor cocktail (Sigma, USA) at 1 mg/ml. The lysing buffer contained 0.5% Triton X-100, 150 mM NaCl,

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15 mM Tris, 1 mM CaCl2 , and MgCl2 (Tsai et al., 1997). The tissues were then homogenized with a tissue homogenizer (Heidolph, Germany). The homogenates were further incubated in ice for 30 min, centrifuged at 1400 × g for 10 min, and the supernatants containing the cytokines were then collected and stored at −20 ◦ C for further analysis.

2.2.2. Protein analysis assay The total protein concentrations in the lung and lymph node tissue homogenates were determined using the Biuret method (Biolab SA, France). This is a colorimetric method, in which absorbance is proportional to the concentration of the protein in the homogenate. Cytokine concentration was presented as pg of cytokine per g of total protein.

2.2.3. NGAL and IGF-1 measurements NGAL or IGF-1 concentrations in sheep lymph nodes, lung homogenates, and serum samples, were measured using an ELISA kit (Cusabio Biotech Co., China). This kit had been pre-coated with an antibody specific to NGAL or IGF-1. Standard, or samples, were added to the appropriate microtiter plate wells, with a horseradish peroxidase (HRP) conjugated antibody preparation, specifically for NGAL or IGF-1, and incubated for 2 h at 37 ◦ C. Then, substrate solutions A and B were mixed and added to each well. Only those wells that contained NGAL, or IGF-1, and those that reacted to an HRP-conjugated antibody, exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured. The concentrations of NGAL, or IGF-1, in the samples, were then determined by comparing the O.D. of the samples to a standard curve at 450 nm.

2.2.4. TNF-˛ and IL-10 measurement TNF-␣ and IL-10 concentrations in the sheep lymph nodes, and lung homogenates and serum samples, were measured using the competitive inhibition enzyme immunoassay technique “ELISA kit” (Cusabio Biotech Co., China). Antibodies, specific to either TNF-␣ or IL-10, were pre-coated onto microtiter plates. Standard, or samples, were added to the appropriate microtiter plate wells, with biotin-conjugated TNF-␣ or IL-10, and incubated for 1 h at 37 ◦ C. Then, Avidin conjugated to HRP was added to each microtiter plate well and incubated for 30 min at 37 ◦ C. The substrate solutions were then added to the wells and incubated for 15 min at 37 ◦ C and color developing was measured by an ELISA plate reader ELx800 (Bio Tex Instruments Inc., Winooski, VT, USA) at 450 nm.

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3. Results 3.1. Identification and confirmation of M. haemolytica isolates One hundred and fifty pneumonic lungs specimens that showed a typical gross lesion of M. haemolytica infection were taken for bacteriological examination (Fig. 1). A total number of 58 (38.7%) out of the 150 lung tissue samples from the sheep were identified as being of M. haemolytica. The isolates of M. haemolytica were also confirmed by a PCR technique, using a PHSSA primer, and revealed a product size of 325 bp (Fig. 2). Nuclease free water that was used as a negative control did not show any amplification band. The sequencing of the PCR product results was obtained from Sequetech, USA, and were compared and interpreted by referring to the NCBI-Blast GenBank (www.ncbi.nlm.nih.gov/BLAST/). The sequence that was compared with the NCBI-Blast Bank matched 99% with the genome of the M. haemolytica strains; PH278, PH8, PH2, PH284, and PH388. Sequence 2 matched 99% with the genome of the M. haemolytica serotype 2, and with the M. haemolytica serotype-1 specific antigen.

Fig. 1. Mannheimia haemolytica affected lung of 6-month old lamb. The right apical lobe showed the consolidated portion of the affected lung (A). The margin of the lesion (B) and health lung portion of lung were also presented (C).

2.3. Statistical analysis Statistical analyses were performed by using a Student’s t-test. The difference was considered significant at a value of (P < 0.05).

Fig. 2. The electrophoresis analysis (2% agarose gel) of the PCR products of M. haemolytica, as amplified by the specific primers from the SSA gene. Lane M = 100 bp DNA marker (Promega Corp, Madison, USA); Lane 1 = positive control of M. haemolytica (NCTC #10627); Lanes 2–7 = M. haemolytica positive samples; Lane 8 = negative control.

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3.2. Inflammatory parameter levels in M. haemolytica affected sheep Seven animals out of 58 that showed a typical gross lesion of M. haemolytica infection, were included in the analysis of the immune parameters. 3.2.1. Detection of NGAL The NAGL levels in the serum of the affected sheep had higher levels of NAGL production than the healthy ones (Fig. 3A). The affected lymph nodes had a similar NAGL production when compared to the lymph nodes of the healthy sheep (Fig. 3B). In the sheep lung tissue homogenate, the highest level of NAGL production was demonstrated in the center of the consolidated part when compared to the other lung tissue homogenates (Fig. 3C). The lungs of the affected sheep that appeared grossly healthy also had higher levels of NGAL production when compared to the margins of the consolidated lesions and the healthy non-infected sheep lung tissue homogenates. 3.2.2. Detection of IGF-1 The IGF-1 distribution followed a similar trend to the NGAL levels detected in the serum and lymph nodes. IGF-1 levels in the serum of the affected sheep had a higher level of IGF-1 production than in the healthy ones (Fig. 4A). However, the affected lymph nodes had a similar IGF-1 production when compared to the lymph nodes of the healthy sheep (Fig. 4B). In the sheep’s lung tissue homogenates, the highest level of IGF-1 production was demonstrated in the center of the consolidated part, and the part appeared grossly unaffected when compared to the margin lesions and the healthy non-infected sheep’s lung tissue homogenate (Fig. 4C). The lung tissue homogenates of the margin lesions had a higher level of IGF-1 production than in the lung tissue homogenate obtained from the healthy non-infected animals. 3.2.3. Detection of TNF-˛ and IL-10 A significant difference was observed in the level of TNF-␣ production in the serum of the affected sheep when compared to the healthy ones (Fig. 5A). Similarly, the lymph nodes of the affected sheep had a higher level of TNF-␣ when compared to the lymph nodes of the healthy sheep (Fig. 5B). In the lung tissue homogenates, the highest level of TNF-␣ production was demonstrated in the center of the consolidated part when compared to the lesion’s edge, as well as when compared to the grossly healthy portion of the same M. haemolytica affected lungs (Fig. 5C). The lungs of the affected sheep that appeared grossly healthy had a higher level of TNF-␣ production than in the lung tissue homogenate that was obtained from the healthy non-infected animals (Fig. 5C). IL-10 distribution followed a similar trend to the TNF-␣ levels that were detected in the different tissues of homogenate and serum. The results are represented in (Fig. 6). 4. Discussion To our knowledge, this is the first study that has closely described the distribution of key inflammatory parameters

in the tissue homogenate of M. haemolytica infected lungs in sheep. The data has proposed, for the first time, an association of NGAL and IGF-1 in the M. haemolytica infection in sheep. M. haemolytica causes pneumonia in ruminants that begin as an inflammatory response in the bronchioles and alveoli of the lung and then resulting in the consolidation of the lung tissues (Aulik et al., 2010). When a certain threshold dose of microorganisms, host susceptibility, and non-specific defense mechanisms are breached, the lung defense mechanisms are deteriorated, allowing for the disease to occur (Angen et al., 2002). Studying innate and adaptive immune reaction to infection plays an important role in enhancing our knowledge in order to improve an antibacterial response. We have shown, for the first time, an increase of NGAL levels in a sheep’s lungs in response to an infection with M. haemolytica. The NAGL level in the serum and the lung tissue homogenate of affected sheep had a higher level of NAGL production than in the healthy ones. However, the affected lymph nodes had a similar NAGL production when compared to the lymph nodes of healthy sheep. NGAL protein was expressed by many cells and tissues, including neutrophils, monocytes, renal tubules cells, liver, and lungs (Daure et al., 2013). NGAL is now considered to be a pivotal component of the innate immune response to bacterial infection. In the innate immune response, NGAL was secreted by Toll-like receptors after a ligation with bacteria. NGAL has the ability to arrest bacterial growth by sequestrating the iron-laden siderophores that are needed to scavenge iron and consequently thus promoting bacterial growth (Flo et al., 2004). Previous studies have demonstrated the role of NGAL protein in many bacterial infections, such as E. coli, Klebsiella and Salmonella in mouse models, as well as in a M. tuberculosis infection in humans (Chan et al., 2009; Martineau et al., 2007; Nairz et al., 2008; Wu et al., 2010). To our knowledge, there is no report about the NGAL expression in sheep lungs. However, in agreement with our result, a previous study on mice models has shown that E. coli induces a strong expression of NGAL in the epithelial cell of the respiratory tract, while the lack of an NGAL expression leads to an increased morbidity and mortality in the infected mice (Wu et al., 2010). Additionally, it has been reported that NGAL protects against the airway inflammation of allergic asthma in mouse modules (Dittrich et al., 2010). This data supports the hypothesis that NGAL is useful in protecting sheep against an infection with M. haemolytica. Recently, many studies have focused on the role of NGAL as a promising biomarker in humans and dogs with an acute kidney injury (Daure et al., 2013). Therefore, this current study has shown that the differential production of NGAL in M. haemolytica infected sheep has the potential to be employed as a diagnostic marker for a lung infection with M. haemolytica. The role of IGF-1 in M. haemolytica infections has never been investigated in sheep. The alveolar macrophages of the affected lungs were the main source of IGF-1 (Rom et al., 1988). The concentration of IGF-1 in the serum of the affected animals in the current work was markedly higher than that that was obtained from the healthy animals. The lungs of the healthy animals also appear to have

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Fig. 3. NGAL levels (pg/g total protein in the sample) in affected animals (n = 7) and healthy animals (n = 7), in (a) serum, (b) lymph node, and (c) lung homogenates. The serum and lymph nodes were collected from healthy non-infected sheep and from M. haemolytica infected sheep (affected). M. haemolytica affected lung tissues were separated according to the pathological changes in normal tissues that had no pathological changes, tissues from the margin of consolidated parts of the lesion, and from the consolidated portion of the M. haemolytica affected lung. Lung tissues from the control sheep that were not infected with M. haemolytica were also analyzed (healthy tissues). Each value represents a mean value ± S.E.M. Different letters indicate a significant difference between the groups (P < 0.05).

a lower IGF-1 production when compared to the affected animals. The pulmonary parenchyma and airways must be remodeled and repaired following a microbial infection and following injury. Repair and fibrotic processes are crucially dependent on growth factors for fibroblasts and epithelial cells, including a platelet derived growth factor (PDGF), transforming the growth factors of alpha and beta (TGF-␣ and TGF-␤), and IGF (Madtes et al., 1999). IGF-1 is a profibrogenic mediator, acting as a potent mitogen and

stimulator of collagen synthesis by fibroblasts (Allen et al., 1998). It is mainly secreted by alveolar macrophages in the lung (Rom et al., 1988), but it can also be expressed by other cell types, such as fibroblasts and epithelial or endothelial cells (Homma et al., 1995). In sheep, IGF-1 most likely plays a role in the epithelial cell repair of the lungs following an injury as in the fibro-proliferative changes in fibrotic lung diseases (Chang et al., 1988; Francis et al., 1989). It seems that lung tissue homogenates from infected animals had an

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Fig. 4. The IGF-1 production (pg/g total protein in the sample) in affected animals (n = 7) and healthy animals (n = 7), in (a) serum, (b) lymph node (Ln), and (c) lung tissue homogenates. The samples were collected from the serum and the lymph nodes that were taken from the control non-infected healthy sheep and the affected samples obtained from the M. haemolytica infected sheep. For lung samples, the healthy group had a tissue homogenate that was obtained from a normal sheep’s lung. Affected tissues were divided into normal tissues that had no pathological changes, tissues from the margin of the consolidated part of the lesion, and from the consolidated portion of the M. haemolytica affected lung. Each value represents a mean value ± S.E.M. Different letters indicate a significant difference between the groups (P < 0.05).

interesting differential production of IGF-1. That is worth further investigation, for this cytokine role in the case of pneumonia is caused by M. haemolytica. An analysis of an M. haemolytica induced cytokines profile would help to clarify some of the immune pathogenesis and immune modulators in pneumonia induced by M. haemolytica. Previous findings have pointed to an association between lung pathology and an increased pulmonary expression of inflammatory cytokines in bovine pneumonic mannheimiosis (Caswell et al., 1998). In this

study, the proinflammatory cytokines that pointed to a Th1 response were represented by a measurement of the levels of TNF-␣, while the anti-inflammatory cytokines response Th2 was assessed through an IL-10 measurement. In most mammalian models, TNF-␣, and IL-8, are the central components of a complex cytokine network that initiates, amplifies, and sustains the inflammatory response in tissue (Malazdrewich et al., 2001, #37; Redondo et al., 2011, #3). Similarly, differences between the inflammatory changes at the center of the consolidated

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Fig. 5. TNF-␣ production (pg/g of total protein in the sample) in affected animals (n = 7) and healthy animals (n = 7), in (a) serum, (b) lymph node (Ln), and (c) lung tissue homogenates. The healthy group, from their serum and lymph nodes, represents samples that were taken from the non-infected control sheep. The affected groups were obtained from M. haemolytica infected sheep. For lung tissue homogenates, samples from the affected animals were collected from the normal tissues that had no pathological changes, the margins of the consolidated part of M. haemolytica affected lung tissues, and from the consolidated portion of the affected lung. Each value represents a mean value ± S.E.M. Different letters indicate a significant difference between the groups (P < 0.05).

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Fig. 6. IL-10 production (pg/g total protein in the sample) in affected animals (n = 7) and healthy animals (n = 7), in (a) serum, (b) tissue homogenates of lymph node (Ln), and (c) lung. Serum and lymph nodes samples were collected from control non-infected sheep (healthy) and the naturally M. haemolytica infected sheep (affected). Lung tissues from healthy non-infected sheep (control), as well as from the M. haemolytica affected lung tissues that had no pathological changes (normal), the margin of the consolidated part, and from the consolidated portion of the affected lung, were analyzed. Each value represents a mean value ± S.E.M. Different letters indicate a significant difference between the groups (P < 0.05).

part and the lesion margins were demonstrated. The lungs of the affected animals, although appearing to be grossly healthy, had a high level of TNF-␣ when compared to the healthy lungs that were obtained from the non-affected animals. Significant increments in the concentrations of

cytokine levels of TNF-␣ were also detected systematically in the serum and in the regional lymph nodes, especially when compared to the healthy animals. Similar results were also detected for IL-10 which is considered to be an anti-inflammatory cytokine. A provoked production

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of cytokines, for both TNF-␣ and IL-10 indicates that the immune response that is activated by M. haemolytica induces both cell mediated and humoral immunity. The results obtained from this research support previous studies in that multiple cytokines contribute to pneumonia that is induced by M. haemolytica (Malazdrewich et al., 2004). TNF-␣ is a proinflammatory cytokine and its expression is usually elevated in the early stages of pneumonia when induced by M. haemolytica (Malazdrewich et al., 2001, #37; Lafleur et al., 2001, #26; Moore et al., 2001, #40; Redondo et al., 2011, #3). On other hand, IL-10 is a regulatory antiinflammatory cytokine, and it is expected to play a role in the resolving stage of pneumonia. The principal routine function of IL-10 appears to limit and ultimately terminate inflammatory responses. It is recognized for its ability to inhibit activation, together with the effector functions of T cells, monocytes, and macrophages, and to regulate the growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, granulocytes, and endothelial cells (Moore et al., 2001). In addition, it has, predominantly, antiinflammatory and immunosuppressive down regulating effects on both the innate and adaptive immune responses, through the suppression of the antigen-presenting functions of macrophages, which in turn, inhibit the effective generation and/or maintenance of antigen-specific Th1 cells (Ozbey et al., 2004). Thus, during an organization of the inflammatory response, as induced by M. haemolytica, the immune system is also seen to be busy in the remolding of the damaged tissue. High IL-10 levels, detected systemically, as well as locally, in the current study, were similar to what was reported by Singh et al. (2011). In the present study, M. haemolytica was isolated from consolidated lungs, and it was identified by using PCR and DNA sequences in addition to conventional tests. All isolates were positive by both culture and PCR. The isolation rate of M. haemolytica was 38.7%, which is similar to the findings of Leite et al. (2004). However, this result was higher than another local study that showed an isolation rate close to 24%. This would indicate that an infection with this type of bacteria is a growing problem in Jordanian flocks (Hawari et al., 2008). It is obvious that the sequence of the (SSA) gene matches 99% with the genome of M. haemolytica. The sequencing of the PCR product complements the PCR results for M. haemolytica isolates. In summary, the current work has demonstrated an association between the pulmonary expression of some inflammatory parameters and those of lung pathology. Similar to previous reports, the distribution of TNF-␣ in affected sheep relates this cytokine with some pathogenic features at the onset disease (Malazdrewich et al., 2004; Redondo et al., 2011). The consolidate tissues represent the earliest lesions in the disease progression, thus TNF-␣ accumulation in this damaged portion can be related to the onset of the disease (Cutlip et al., 1998; Ramirez-Romero and Brogden, 1995). The NGAL production might be introduced as a useful parameter in protecting sheep against an infection with M. haemolytica. At the same time, the production of IGF-1, and IL-10, are required for the efficient renovation of the epithelial injury.

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