INTRODUCTION

Haemorrhagic Septicaemia (HS) caused by mainly Pasteurella multocida manifest often as respiratory infection with characteristic of per-acute signs in various species of animals. It is one of the most common diseases of cattle, goats and sheep in tropical countries and has a major of economic importance and welfare implications worldwide. Outbreaks of this disease usually relate to high mortality and great economic alterations to the ruminant industry (Gilmour et al., 1991; Brogden et al., 1998; Odugbo et al., 2003). This is evidenced by the global attention and the political debate of this disease particularly in temperate areas including Malaysia. The organism is important of primary or secondary animal pathogen for over a century and is becoming important as human pathogen leading to a disease entity termed as pasteurellosis. The organisms usually act as secondary invaders in animals with concurrent diseases or suffering from debilitating stressful conditions (Benirschke et al., 1978). The serotype B has been recovered from HS-affected animals in Southern Europe, Central and South America, the Middle East and Asia, including Malaysia. The infection causes substantial morbidity and mortality in cattle, buffaloes, sheep and goats (Bain et al., 1982). In Malaysia, the stressful condition is during the raining season where most outbreaks occurred (Saharee et al., 1993).

Although, in Malaysia Haemorrhagic Septicaemia (HS) does appear to receive the attention it deserves, the disease, however, is still threatening the large ruminant sector resulting economic losses in the production of milk, meat and the by-products such as hide for the leather industry. According to the National Immunization Plan (NIP), killed whole cell vaccines have been used in Malaysia to control the Haemorrhagic septicaemia. These vaccines have not been nationally licensed due to the defects that imply shorter duration of immunity and swelling at the site of inoculation. However, previous studies have hypothetically proposed that the capsular antigens; Lipopolysaccharide (LPS) or LPS-protein complex and the Outer Membrane Proteins (OMPs), including the iron-regulated OMPs might be useful as an effective immunogens for sero-groups B and E (Carter and de Alwis, 1989). However, the mechanisms of these immunogens remain largely obscure and to improve strategies for the overall understanding of these components, it is desirable to establish a base line data in relation to the blood picture of these immunogens in the actual host. Thus, the objective of this study was to determine the changes in the blood parameters of cattle following infection by live P. multocida B: 2 and inoculation with the bacterial OMP and LPS.

MATERIALS AND METHODS

Animals: Eight clinically healthy calves of approximately 12 months of age and weighing 150±50 kg were selected from a farm. Nasal swabs were collected immediately after selection and every 3 days for at least 2 weeks to ensure that the calves were free of P. multocida prior to the start of the experiment. The animals were then transported by road to the Animal Experimental Unit (AEU). Upon arrival at the AEU, anthelmintic (Ivermectin) was administered subcutaneously at the rate of 1 mL 50 kg^-1 b.wt. to control internal parasitism which has been shown to influence disease development. The animals were then divided equally into 4 groups, kept in separate rooms and fed with cut grass and supplemented feed at the rate of 1 kg day^-1.

Inoculum: A total of three types inocula were used throughout the experiments: (1) Wild-type P. multocida B: 2 isolated from a previous outbreak of HS in the state of Kelantan, Malaysia; (2) The Lipopolysaccharide (LPS) extracted from P. multocida B: 2 and (3) Outer Membrane Proteins (OMP) extracted from P. multocida type B (Table 1). The inocula (LPS and OMP) were extracted according to the method described by Abdullah et al. (2013). Isolates of P. multocida type B were collected from freeze-dried stock cultures and grown on blood agar for 24 h. McFarland method was used to determine the concentration of the inocula.

Table 1:	Experimental design
*N/A: Not applicable, aLipopolysaccharide and bOuter membrane protein

Experimental procedure: At the start of the experiment, the heifers were divided into 4 groups of 2 cows in each group. Cows of group 1 served as control group and were inoculated intramuscularly with 10 mL of sterile Phosphate Buffered Saline (PBS). Cows of group 2 were inoculated intramuscularly with 10 mL of 10¹² Colony Forming Unit (CFU) of wild-type P. multocida. Cows of group 3 received intravenously the inoculum containing 10 mL of LPS broth extract. Cows of group 4 received intramuscularly the inoculum containing 10 mL of OMP broth extract (Table 1). Following intramuscular/intravenous challenge, all animals were observed for 48 h for signs of respiratory infection and mortality pattern before the surviving animals were euthanized after 48 h post-inoculation. Serum samples were collected from all challenged and non-challenged animals via jugular venipuncture at 3 h intervals. The entire respiratory tracts were monitored and the extent of the damage of the visceral organs with emphasis on lung lesions was determined according to the method described by Gilmour et al. (1990).

Blood analysis
Haematology: Hematocrit, total solids and white blood cell counts were conducted using the Cell-Dyn 3700 Automatic Analyzer (Vet Package, Abbot Diagnostic, 2007) conducted at the Hematology and Biochemistry Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Pack Cell Volume (PCV) was determined by micro-haematocrit technique. Blood smears were stained with Wright Stain (Sigma^®) for differential counting of leucocytes which is the percentage of lymphocytes, monocytes, basophils, eosinophils and neutrophils. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) were analyzed using ST Art 4^® (Diagnostic Stago, France).

Serum biochemistry: Sera were processed by automated chemistry analyzer (HITACHI 902 Automatic Analyzer^®, Japan) for biochemistry parameters. The analyzed parameters include sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphate (P), Blood Urea Nitrogen (BUN), creatinine (Crea), glucose, Total Bilirubin (T. Bill), Total Protein (TP), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST), Creatinine Kinase (CK), Albumin (Alb), Gamma-glutmyltransferase (GGT) and globulin and albumin ratio (A:G).

Statistical methods: The mean values obtained from haematological and biochemical assays of samples from infected animals with Pasteurella multocida type B and its immunogens; LPS and OMP were compared with data from the control group using the Tukey-test. Differences were considered to be statistically significant with values of p<0.05.

RESULTS

Blood and biochemistry: Haematologically, the characteristics of tested parameters in the different groups involved in the study are summarized in Table 2. There were significant differences in the mean values of Prothrombin Time (PT), Partial Thromboplastin Time (PTT) and thrombocyte levels in the challenged groups 2-4 compared to the control group (Table 1). Mean PT was significantly higher in group 2-4 with mean levels 25.300±4.623; 23.292±1.780; 22.250±1.512, respectively than the negative control group. For Partial Thromboplastin Time (PTT), only group 3 (30.327±1.543) showed a significant 1 time increased compared to the negative control (Table 2). Similarly, mean levels of thrombocyte were significantly decreased in group 2-4 (239.571±75.681; 296.029±79.48; 335.49±125.20, respectively) than control group with mean values of 404.50±96.763 (Table 2).

Among the challenged groups, the animals received the inoculum containing live Pasteurella multocida showed significant differences in PCV and leukocyte counts where this group showed no significant differences in RBC and Hemoglobin counts compared to control group. Both groups 3 and 4 inoculated with immunogens (LPS and OMP) did not show significant difference in leucocyte counts and white blood cells. There were relatively little effect of immunogens LPS and OMP broth extract compared to live P. multocida B: 2 on all investigated hematological parameters (Table 2).

There was no significant differences in the serum levels of Gamma-glytamyltranspeptidase (GGT) and almost all tested electrolytes; Na, Cl and K (Table 3). In contrast, all challenged groups showed significant decrease in the levels of calcium ion for group 2-4 with mean values of 1.851±0.319, 2.185±0.176 and 2.260±0.141 mmol L^-1, respectively. Similarly, there was significant decrease in the mean concentration of Alanine Transaminase (ALT) for group 2 (20.607±1.639 U L^-1), group 3 (22.067±1.617 U L^-1) and group 4 (40.327±8.351 U L^-1). Moreover, there was no significant difference in Alkaline Phosphatase (ALP) for group 2 (149.857±17.327 U L^-1) but a significant decrease for groups 3 (114.147±20.901 U L^-1) and group 4 (128.971±17.068 U L^-1).

Table 2:	Blood haematology changes in cattle inoculated with various immunogens of P. multocida B: 2
aRed blood cells, bHaemoglobin, cPacked cell volume, dWhite blood cells, eBanded neutrophils, fSegmented neutrophils, gBasophils, hLymphocytes, IMonocytes, jEosinophils, kProthrombin timem, lActivated partial thromboplastin time. *Significant value p<0.05. Comparison between immunogens groups and negative control group

Table 3:	Blood serum biochemistry in calves inoculated with different immunogens of P. multocida B: 2
aAlanine transaminase, bAlkaline phosphatase, cAspartate transaminase, dCreatine kinase, eGlobulin, fSodium, gPotassium, hChloride, iCalcium, jGamma-glutamyl transpeptidase, kAlbumin/globulin ratio. *Significant value p<0.05. Comparison between immunogens groups and negative control group

In Aspartate Transaminase (AST), only group 4 (188.579±45.581 U L^-1) showed significant increase in the mean concentration where groups 2-3 showed no differences with mean values of (79.250±9.940 U L^-1) and (68.868±3.784 U L^-1), respectively. In total protein levels, group 3 (81.547±2.235 g L^-1) and 4 (87.921±3.619 g L^-1) showed no significant difference but group 2 (55.079±7.578 g L^-1) showed a significant increase in the level of total protein compared to the negative control (Table 3). Similarly, groups 3 (48.871±1.770 g L^-1) and 4 (51.735±2.192 g L^-1) showed no significant difference in the globulin level but group 2 (32.843±5.194 g L^-1) showed a significant decrease in the level of globulin. There was no significant difference in Creatine Kinase (CK) level in groups 2 (296.643±74.524 U L^-1) and 3 (244.912±58.513 U L^-1) but a significant 16 fold increase in group 4 (4114.500±1472.42 U L^-1) (Table 3).