Abstract: In this study, haematological traits, lysozyme concentration and T lymphocyte subpopulation as markers of innate immunity were detected and compared among Landrace, Large White and a chinese indigenous breed, Songliao Black pig. The animals were of the same age and kept under the same environmental conditions to reduce non-genetic variation in immune traits; they were all apparently healthy and were vaccinated by CSF live vaccine at 21 days of age. Except LY%, MO%, CD4^�CD8^�% and RDW, the other detected immune traits were significant difference between before (20 days) and after vaccination (35 day). While the values of MCV, MCH, MCHC, MO%, CD4⁺CD8⁺% and CD4⁺CD8⁺/CD4^�CD8⁺ values decreased after vaccination, the others increased. The values of WBC, GR, LY, MO, CD4⁺CD8^�% and CD4⁺CD8⁺% were significant difference among Large White, Landrace and Songliao Black pig (p<0.05). Our analysis confirms that Songliao Black pig has better innate immune level than Landrace and Large White. The animal resource population was suggested to be appropriate to investigate further the QTL and genes contributing to differences on these innate immune traits in pigs.

INTRODUCTION

Animal disease resistance is the result of interaction of innate (non-specific) and specific immunity and both of them are under genetic control (Muller and Brem, 1991). The innate immune system comprises the cells and mechanisms that defend the host from infection by other organisms, in a non-specific manner and is general resistance to disease; it reflects the overall defense capacity against diseases (Alberts et al., 2002). The improvement of the general resistance to disease for enhancing the overall immune function and identification of these variants of immune traits may be suitable for the genetic improvement through selection (Lamont, 1998). However, innate immune traits and disease resistance also impact upon animal performance. Genetically improving disease resistance in pigs may or may not impinge on performance, depending upon the pathogen and the nature of the infectious challenge (Colditz, 2002). Chinese breed Meishan pig and western breed Large Whites pig have been appeared to differ in resistance to a wide variety of pathogens and also differ in various innate immune traits (Clapperton et al., 2005; Sutherland et al., 2005). Genetic component to variation in several innate immune traits has been reported in pigs (Edfors-Lilja et al., 1994; Clapperton et al., 2005) and some Quantitative Trait Llocus (QTLs) also has been identified in pigs (Edfors-Lilja et al., 1998; Gong et al., 2010; Reiner et al., 2007; Wattrang et al., 2005).

Haematological traits are essential parameters for evaluating the health status of individual animals and herds (Gong et al., 2010). Lysozyme concentration in tissue fluids also reflect the number of activated macrophages (Hyyppa et al., 1989). T-lymphocytes represent an important cell population of the immune system and perform a wide array of functions in immune regulation, inflammation and protective immune responses (Summerfield et al., 1996). Identifying the variants in these innate immune traits would be a helpful step towards exploring any link to disease resistance capacity and improve a measure to advance general health in the pig. In order to investigate the differences of innate immune traits on different pig breeds and further build an animal resource population for QTL mapping studies, we firstly chose and detected the haematological traits, lysozyme concentration and T lymphocyte subpopulation which represent different aspects of innate immunity. At last, it also further compared the haematological traits, lysozyme concentration and T lymphocyte subpopulation before and after vaccination among Landrace, Large White and a Chinese indigenous breed, Songliao Black pig.

MATERIALS AND METHODS

Animals: The animals consisted of 338 piglets distributed in three pig breeds including Large White (169), Landrace (84) and Songliao Black pig (85). All pigs were raised under the standard indoor condition in 2007 and 2008 at the experimental farm of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. All pigs were apparently healthy and vaccinated with Classical Swine Fever (CSF) live vaccine at 21 days of age.

Blood samples: Two measurements were performed on 20 days old and 35 days old piglets respectively. For each piglet, 10 mL blood was collected via the external jugular vein. The first blood samples were collected from each piglet one day before the vaccination (20 days ) and two weeks after the vaccination; the second blood samples were collected (35 days). All blood samples were divided into two tubes: the first tube contained EDTA anti-coagulant for the measurement of haematological traits and T lymphocyte subpopulation; the second tube was directly injected into VACUETTE^® Serum Clot Activator tubes for detection for lysozyme concentration.

Measurement of haematological traits and lysozyme concentration in blood: Haematological traits which consist of mainly three components, including leukocyte traits, erythrocyte traits and platelet traits were measured. Total 18 blood parameters include 7 leukocyte traits (White Blood Cell Count (WBC), neutrophilic granulocyte count (GRAN) and percentage (GR%), lymphocyte count (LYMF) and percentage (LY%), monocytes count (MONO) and percentage (MO%)), 7 erythrocyte traits (Red Blood Cell (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) and Red Blood Cell volume distribution width (RDW)), 4 platelet traits (blood Platelet counts (PLT), Mean Platelet Volume (MPV), Platelet Distribution Width (PDW) and Plateletocrit (PCT). All these blood routine parameters were measured by MEK-6318K type full automatic Hematology Analyzer (Nihon Kohden, Japan). Lysozyme Concentration (LSZ) in serum was measured by turbidimetric analysis using the lysozyme testing kit (Jiancheng Institute of Biotechnology, Nanjing, China).

Measurement of T lymphocyte subpopulation in blood: T lymphocyte subpopulations (CD4⁺CD8⁺%, CD4⁺CD8^�%, CD4^�CD8⁺% and CD4^�CD8^�%) were detected by EPICS Flow Cytometer (Beckman-Coulter Company, USA) with an argon laser with an excitation wavelength of 488 nm and using FITC-CD4/PE-CD8 monoclonal antibody (Serotech Ltd., England). The percentage of monoclonal antibody CD8/CD4 cells was obtained for each sample tested and the purity of the separated fractions was ≥98%.

RESULTS AND DISCUSSION

Except LY%, MO% and RDW, the other detected haematological traits were significant difference between before and after vaccination by paired t-test (Table 1). The values of MCV, MCH, MCHC and MO% decreased and those of the other haematological traits increased after vaccination.

Table 1:	Comparative analysis of haematological traits, T lymphocyte subpopulations, lysozyme concentration before and after vaccination
1WBC, White Blood Cell count; GRAN, neutrophilic granulocyte count; GR% neutrophilic granulocyte count percentage; LYMF, lymphocyte count; LY% lymphocyte count percentage; MONO, Monocytes count; MO%, percentage; RBC, Red Blood Cell; HGB, Hemoglobin; HCT, Hematocrit; MCV, Mean Corpuscular Volume; MCH, Mean Corpuscular Hemoglobin; MCHC, Mean Corpuscular Hemoglobin Concentration; RDW, Red blood cell volume distribution width; PLT, blood platelet counts; MPV, Mean Platelet Volume; PDW, Platelet Distribution Width; PCT, Plateletocrit; CD4+CD4�CD8+CD8� T lymphocyte subpopulation; LSZ, Lysozyme concentration

The values of WBC, GR%, LY and MO were significant difference among Large White, Landrace and Songliao Black pig (Table 2). The breed and age of pig are important factors influencing immune response to various infectious challenges (Sutherland et al., 2005). In a study for two Australian pig breeds, WBC and CD4⁺. T lymphocyte count of Large White are higher than Duroc after leptospira vaccination (Nguyen et al., 1998). Moreover, compared with the Large White, Meishan pig has higher neutrophils and monocyte counts and lower lymphocyte counts (Clapperton et al., 2005). In this study, WBC, PLT, GR% and GRAN of Chinese indigenous breed Songliao Black pig were higher increased after vaccinated with CSF live vaccine than western pig breed Landrace and Large White, this is similar to Meishan pig.

Lysozyme, also called bacteriolysin is found in saliva and has been shown to have an antibody-independent defense function (Hyyppa et al., 1989). Lysozyme concentration in serum was significant difference between 20th and 35th day (p = 0.0017) and the value of LSZ was also increased after vaccination (Table 1). Among three pig breeds, the value of LSZ was significant difference (p<0.05) and Songliao Black pig has higher level than Landrace and Large White (Table 2). Lysozyme was synthesized and released by macrophages, the elevation in lysozyme levels consequent upon the growth of antigenic tumors may be a reflection of an increase in the number of macrophages (Currie and Eccles, 1976). The results suggest that Songliao Black pig have much more macrophages numbers than landrace, Large White.

Table 2:	Multiple comparative analysis for difference value of haematological traits, lymphocyte subpopulations and lysozyme concentration in three pig breeds
a.bSigned by small letters differ significantly at p<0.05

T lymphocytes represent an important cell population of the immune system. The detection of T lymphocyte subpopulation by Flow Cytometer was shown in Fig. 1. Three of four T lymphocytes subpopulation except CD4^�CD8^� were very significantly different before and after vaccination (p<0.01) and the percentage of CD4⁺CD8⁺, CD4^�CD8⁺ and CD4^�CD8^� T lymphocyte increased (Table 1).

In addition, the percentage of three T lymphocytes except CD4^�CD8⁺ was also significantly different among three pig breeds (Table 2). The variation in the percentage of CD4⁺CD8⁺ T lymphocyte is confirmed increasing with the age of the animals (Summerfield et al., 1996). The extrathymic CD4⁺CD8⁺ T lymphocyte subpopulation of swine contains MHC class II restricted antigen-specific memory T helper cells and this CD4⁺CD8⁺ T lymphocyte subpopulation could be characterized as mature T lymphocytes morphologically and phenotypically (Pescrovitz et al., 1994). Lohse et al. (2006) also reported that increase of CD4⁺CD8⁺ lymphocyte may contribute to host resistance against infectious diseases. Therefore, CD4⁺CD8⁺ T lymphocyte is an important reflection of immune status in pigs. In this study, the percentage of CD4⁺CD8⁺ in Songliao Black pig has higher level than Large White but is lower level than that of Landrace (Table 2). The results suggested Songliao Black pig have better immune capacity in three pig breeds.

In this experiment, various innate immune parameters detected differences among three pig breeds and all parameters belong to quantitative traits. Significant differences of immune traits before and after vaccination reflect whether immune responses or the strength of its levels are induced.

Fig. 1:	Alloantigen-specific response of porcine T lymphocyte subpopulations and CD8 versus CD4 expression of the un-separated T lymphocyte was detected by Flow Cytometer. The fluorescence intensities of CD8 (PE, abscissa) and CD4 (FITC, ordinate) are displayed in two-dimensional contour plot

The compared results may indicate Songliao Black pig has higher immune capacity or disease resistance capacity than other two pig breeds.

Furthermore, the detected results also suggested the pig resource populations can be used for QTL mapping studies to detect genome regions containing genes contributing to differences of these immune traits in the pig.

CONCLUSION

We detected differences among Landrace, Large White and Songliao Black pigs in several innate immune traits before and after vaccination. Differences in the measured immune traits could be because the breeds differ in their basal levels of these traits and in their immune capacity. The results indicated the Chinese indigenous breed, Songliao Black pig, may has higher immune capacity or disease resistance than Landrace, Large White. These results also suggested genetic differences in these marker traits were provided evidence for disease resistance capacity in different pig breeds and the resource populations may be suitable for QTL mapping studies to detect genome regions containing genes contributing to differences in these immune traits.

ACKNOWLEDGEMENTS

This research is supported by China Postdoctoral Science Foundation Funded Project (Grand No. 20100470412); National Key Project for Basic Research and Development Plans (Grant No. 2006CB102104) and the Project of Anhui Scientific Technology Plan (Grant No. 07010300136).