Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis

Proteinograma sérico de bezerros com pneumonia induzida pela inoculação intrabronquial de Mannheimia (Pasteurella) haemolytica

Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis

Proteinograma sérico de bezerros com pneumonia induzida pela inoculação intrabronquial de Mannheimia (Pasteurella) haemolytica

J.J. Fagliari^I; D.J. Weiss^II; D. McClenanhan^II; O.A. Evanson^II

^IFaculdade de Ciências Agrárias e Veterinárias da UNESP Via de Acesso Prof. Paulo Donato Castellane, km 5 14884-900 - Jaboticabal, SP

^IIDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, USA

ABSTRACT

Ten healthy 2 to 4-week-old Holstein calves were randomly allotted into control and infected groups. Control calves (n=5) were inoculated intrabronchially with 5ml of Dulbecco's phosphate-buffered saline solution (DPBSS). Infected calves (n=5) were inoculated intrabronchially with 5x10⁹ log-phase Mannheimia haemolytica organisms suspended in 5ml of DPBSS. Blood samples were obtained 15 minutes before and one, two, four and six hours after inoculation. Serum protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Serum concentrations of proteins with molecular weights of 125,000 D (ceruloplasmin), 60,000 D (a ₁-antitrypsin), 45,000 D (haptoglobin), and 40,000 D (acid glycoprotein) were significantly increased in calves with pneumonic pasteurellosis, compared with concentrations in control calves. Results indicate that acute phase proteins increase more rapidly after the onset of inflammation than previously thought. Measurement of serum protein concentrations may be useful in monitoring the progression of the induced pneumonic pasteurellosis in calves.

Keywords: calf, pneumonia, Mannheimia (Pasteurella) haemolytica, serum protein

RESUMO

Foram examinados 10 bezerros da raça Holandesa com duas a quatro semanas de idade, distribuídos aleatoriamente em dois grupos, controle e infectado. Os bezerros do grupo-controle foram inoculados por via intrabronquial com 5ml de solução salina fosfato-tamponada Dulbecco (DPBSS). Os do grupo infectado receberam um inóculo contendo 5x10⁹ unidades formadoras de colônia-UFC de Mannheimia (Pasteurella) haemolytica, suspensas em 5ml de DPBSS. As amostras de sangue foram colhidas 15 minutos antes e uma, duas, quatro e seis horas após a inoculação. O proteinograma sérico foi obtido por eletroforese em gel de acrilamida. As concentrações séricas das proteínas de pesos moleculares 125.000 D (ceruloplasmina), 60.000 D (a₁-antitripsina), 45.000 D (haptoglobina) e 40.000 D (glicoproteína ácida) foram significativamente maiores em bezerros infectados do que nos do grupo-controle. Os resultados indicam que as concentrações das proteínas de fase aguda se elevaram mais rapidamente que o previamente imaginado. O proteinograma sérico pode ser útil no monitoramento da evolução da pneumonia de bezerros causada por M. haemolytica.

Palavras-chave: bezerro, pneumonia, Mannheimia (Pasteurella) haemolytica, proteínas séricas

INTRODUÇÃO

Colonization of the bronchioles and alveoli of cattle by Mannheimia (Pasteurella) haemolytica organisms results in severe acute lung injury frequently leading to respiratory failure and death. The acute stage of bovine pneumonic pasteurellosis is characterized by severe endothelial damage in alveolar septal capillaries that results in flooding of alveoli with plasma and erythrocytes. When pneumonic pasteurellosis is experimentally induced by administering M. haemolytica into a terminal bronchus, endothelial damage is detected within one to two hours (Ackermann et al., 1996).

Because pneumonic pasteurellosis is an inflammatory condition, it likely provokes changes in serum concentration of acute phase proteins (Heindrich et al., 1990; Kent, 1992) that could be identified by means of electrophoresis (Kent, 1992). If so, then measuring changes acute phase proteins, might be useful in detection of preclinical disease and monitoring progression of pneumonic pasteurellosis. Cellulose acetate and agarose gel electrophoresis have been used to analyze plasma protein concentrations, but these techniques are limited, because they can identify only five to seven groups of proteins (Fagliari et al., 1991). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), on the other hand, can be used to identify more than 20 proteins, can separate proteins present in very low quantities, and can be performed on small plasma samples. The technique has been used to analyze concentrations of plasma or serum proteins (Weber, Osborn, 1969; Fagliari et al., 1998), but has not been previously used to determine changes in serum protein concentrations associated with pneumonic pasteurellosis. The purpose of the study was to determine, by means of SDS-PAGE, the spectrum of serum protein alterations in pneumonic pasteurellosis and the rate at which these changes occur.

MATERIALS AND METHODS

Healthy male Holstein calves, 2- to 4- weeks-old, were obtained from private farms. Calves had been given one dose of colostrum within 12 hours after birth and were observed for at least one week before initiation of the study. Antibody titers to M. haemolytica whole-cell antigens were evaluated by use of an ELISA (Srinand et al., 1996). Titers of calves varied between 1:8 and 1:32. These titers were low, compared with the titer (1:512) for a yearling steer with naturally-developing pneumonic pasteurellosis.

Ten calves were randomly allotted into control and infected groups. Calves were sedated with xylazine hydrochloride (TranquiVed. VEDCO Inc., St Joseph, MO, USA) (0.10mg/kg of body weight, IV) and maintained under light surgical anesthesia by use of sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL, USA) (0.5mg/kg, IV, every 30 to 45 min). An indwelling catheter was placed in the right jugular vein of each calf and a bronchoalveolar lavage catheter with cuff was inserted into the right caudal lung lobe (Rashid et al., 1997). Control calves (n=5) were inoculated intrabronchially with 5ml of Dulbecco's phosphate-buffered saline solution (DPBSS). Mannheimia (Pasteurella) haemolytica-infected calves (n=5) were inoculated intrabronchially with 5x10⁹ log-phase M. haemolytica organisms suspended in 5ml of DPBSS (Rashid et al., 1997). Blood samples were obtained 15 minutes before and one, two, four and six hours after inoculation of saline solution or organisms. Six hours after inoculation of M. haemolytica calves were euthanatized by barbiturate (Nembutal, Abbott Laboratories, Chicago, IL, USA) overdose. The presence of pneumonic lesions in the right caudal lung lobe of infected calves was confirmed by gross observations of lungs and by histologic examination.

Mannheimia (Pasteurella) haemolytica, serotype A1, strain 12298 was isolated from a calf with naturally acquired pneumonic pasteurellosis and cultured according McClenahan et al. (2000). Bacteria were adjusted to 1x10⁹ organisms/ml of sterile DPBSS by use of a Klett Summerson spectrophotometer and a standard colony-forming unit dilution curve.

Serum protein concentration was determined by means of SDS-PAGE (Weber, Osborn, 1969). Gels were stained for five minutes in 200ml of coomassie blue (Sigma Chemical Co., St Louis, MO, USA) and destained in 7% acetic acid until the gel background was completely clear. Concentration of protein fractions was determined by use of computer-assisted videodensitometry (Shimadzu CS 9000. Shimadzu Corp., Kyoto, Japan). Proteins were identified by use of reference markers (Nembutal, Abbott Laboratories, Chicago, IL, USA) with molecular weights of 29,000, 45,000, 66,000, 97,400, 116,000, and 205,000 daltons and by comparison with electrophoretic mobility of purified albumin (Nembutal, Abbott Laboratories, Chicago, IL, USA), transferring (Nembutal, Abbott Laboratories, Chicago, IL, USA), haptoglobin (Nembutal, Abbott Laboratories, Chicago, IL, USA), and a ₁-antitrypsin (Nembutal, Abbott Laboratories, Chicago, IL, USA).

To determine whether serum protein concentrations were significantly different between groups (infected vs control) throughout time, data were analyzed by use of ANOVA for repetead measures followed by F tests. A value of P<0.05 was considered significant. Values are expressed as means ± SEM.

RESULTS

Gross or histologic lesions were not detected in lungs of control calves. Lung sections from M haemolytica-infected calves contained fibrinonecrotic lesions typical of pneumonic pasteurellosis that were restricted to the right caudal lung lobe. Lesions consisted of thick alveolar walls and multifocal fibrin deposits within alveoli, in interlobular areas, and on the lung surface. Inflammatory cells, consisting mostly of neutrophils and erytrocytes, were trapped within the fibrinous exudate. Extensive alveolar septal necrosis was observed. Bacteria were seen in all tissue sections.

Fifteen serum proteins with molecular weights ranging from 18,000 to 168,000 daltons were identified in all 10 calves. Serum concentrations of proteins with molecular weights of 125,000 D (ceruloplasmin), 60,000 D (a ₁-antitrypsin), 45,000 D (haptoglobin), and 40,000 D (acid glycoprotein) were significantly increased in calves with pneumonic pasteurellosis compared with concentrations in control calves (Tab 1). One and two hours after M haemolytica inoculation, the protein with the largest percentage increase (55%) in concentration was a ₁-antitrypsin, compared with baseline values (Fig. 1). Four and six hours after bacteria inoculation, the protein with the largest percentage increase in concentration was acid glycoprotein (441%). The highest percentage increase in concentration of haptoglobin (31%) and ceruloplasmin (104%) were at two hours and four hours after organisms inoculation, respectively.

DISCUSSION AND CONCLUSIONS

In this study, SDS-PAGE allowed to detect 15 distinct protein bands in calf serum, whereas in previous studies (Fagliari et al., 1991) in which cellulose acetate or agarose gel electrophoresis was used, only five or seven protein bands were identified.

In calves with experimentally induced pneumonic pasteurellosis, serum ceruloplasmin, a ₁-antitrypsin, haptoglobin, and acid glycoprotein concentrations were significantly increased one to two hours after M. haemolytica inoculation. These proteins have previously been identified as acute phase proteins (Gruys et al., 1994; Godson et al., 1996). Acute phase proteins are synthesized by the liver in response to inflammatory cytokines, particularly interleukin-6 (Heindrich et al., 1990). Therefore, the increase in serum concentration of acute phase proteins indicated that an inflammatory response had developed in the early stage of experimentally induced pneumonic pasteurellosis, problably due to lung damage (McClenahan et al., 1999; McClenahan et al., 2000).

Results indicate that acute phase proteins increase more rapidly after the onset of inflammation than previously thought. Because of this, it may be possible to use measurements of these proteins to detect infection during the prodromal stages of inflammatory disease. Detection of preclinical disease during an outbreak of pneumonic pasteurellosis or other bacterial disease would permit early isolation and treatment of these calves. In conclusion, measurement of serum protein concentrations may be useful in monitoring the progression of the induced pneumonic pasteurellosis in calves.

Recebido para publicação em 20 de setembro de 2002

Recebido para publicação, após modificações, em 17 de março de 2003

E-mail: [e-mail]

• ACKERMANN, M.R.; KEHRLI, M.R.; BROGDEN, K.A. Passage of CD18-and CD18+ bovine neutrophils into pulmonary alveoli during acute Mannheimia haemolytica pneumonia. Vet. Pathol., v.33, p.639-644, 1996.
• FAGLIARI, J.J.; MCCLENAHAN, D.; EVANSON, A.O. et al. Changes in plasma protein concentrations in ponies with experimentally induced alimentary laminitis. Am. J. Vet. Res., v.59, p.1234-1237, 1998.
• FAGLIARI, J.J.; OKUDA, H.T.; PASSIPIERI, M. et al. Serum protein levels of Guzera cattle in different ages. Arq. Bras. Med. Vet. Zootec., v.43, p.39-60, 1992.
• GODSON, D.L.; CAMPOS, M.; ATTAH-POKU, S.K. et al. Serum hepatoglobin as an indicator of the acute phase response in bovine respiratory disease. Vet. Immunol., v.51, p.277-292, 1996.
• GRUYS, E.; OBWOLO, M.J.; TOUSSAINT, M.J.M. Diagnostic significance of the major acute phase proteins in veterinay clinical chemistry: A review. Vet. Bull., v.64, p.1009-1018, 1994.
• HEINDRICH, P.C.; CASTELL, J.V.; ANDUS, T. Interleukin-6 and the acute phase response. Biochem. J., v.265, p.621-636, 1990.
• KENT, J. Acute phase proteins: their use in veterinary diagnosis. Br. Vet. J., v.148, p.279-282, 1992.
• McCLENAHAN, D.; FAGLIARI, J.J.; EVANSON, A.O. et al. Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis. Am. J. Vet. Res., v.60, p.1307-1311, 1999.
• McCLENAHAN, D.; FAGLIARI, J.J.; EVANSON, A.O. et al. Role of platelet-activating factor in alveolar septal injury associated with experimentally induced pneumonic pasteurellosis in calves. Am. J. Vet. Res., v.61, p.248-254, 2000.
• RASHID, J.; WEISS, D.J.; BACH, R. et al. Role of tissue factor in intra-alveolar fibrin depositon and coagulopathy associated with pneumonic pasteurellosis in cattle. Am. J. Vet. Res., v.58, p.28-33, 1997.
• SRINAND, S.; HSUAN, S.L.; YOO, H.S. et al. Comparative evaluation of antibodies by commercial Mannheimia haemolytica vaccines using solid phase immunoassays. Vet. Microbiol., v.49, p.181-195, 1996.
• WEBER, K.; OSBORN, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., v.214, p.4406-4412, 1969.

Publication Dates

• Publication in this collection
  12 Nov 2003
• Date of issue
  Aug 2003

History

• Accepted
  17 Mar 2003
• Received
  20 Sept 2002