Standardization of Pro-Inflammatory Cytokines (IL-1 & TNF-α) and HaematologicalBiomarker as an Early Assessment of Health Status in Adult Healthy Pigs

The study was aimed to standardize pro-inflammatory cytokines and haematological biomarker level in visually adult healthy swine as an early health check-up scanner. In the present study, total six (6) crossbreed pigs (Yorkshire × Swedish Landrace) with similar age group (3 years) and unisex (male) with average body wt (140 kg) were selected for sampling. Analyzed serum samples revealed that, average values of pro-inflammatory cytokines TNF-α was 242.33± 10.23 ng/ l and IL-1 was (12.16±1.16 ng/l). After analyzed, haematological biomarker showed that, average values of TEC was 6.43±0.14 10 6/μl, Hb was 12.33±0.33 gm/dl, HCT was 37.68±0.5%, PCV was 36.5±1.1%, MCV was 52.66±1.1, MCH was 16±0.8fl, MCHC was 30.83±0.71pg, TLC was 14.75±0.61/μl, neutrophils was 37.2±0.89%, lymphocyte was 54.6±0.62%, monocyte was 2.16±0.34%, eosinophils was 4.86±0.18%, basophils was 1.16±0.18%, platelets was 4.49±0.1 105/μl and total protein was 6.57±0.23 g/dl. In the statistical analysis positive correlation was found between pro-inflammatory cytokines (TNF-α & IL-1β) and routine haematological inflammatory indicators mainly WBC and neutrophils. Lymphocyte showed negative correlation with pro-inflammatory cytokines and other haematological biomarker.

Keywords: Pro-inflammatory cytokines; Indicator; Immune; Poor hygiene; Pig health

Sus scrofa domesticus is homoeothermic animal that have the ability to maintain core body temperature. Extreme fluctuating weather is a major challenge to maintain core body temperature and immune status. In the subtropical climatic condition of India, pigs are mainly habitat under commercial or natural condition in India. However, surrounding hygienic status is major hazard for health status and wellbeing of animals. Poor hygiene level, continuously predisposed to animal for interact to various pathogenic and non-pathogenic micro-organisms throughout entire life. Besides this, some micro-organisms do not clinically affect but they have the ability to produced proinflammatory cytokines and disarrangement of haematological dynamic. These exposures typically respond to their immunological response system (cellular and humeral components) by stimulate the release of pro-inflammatory cytokines and rearrangements of haematological biomarkers (Kim, 2012) [1]. Cytokines such as tumor necrosis factor (TNF-α) is produced by activated macrophages, monocytes, and various other cells. TNF-α is more closely associated with proinflammatory effects (Silva et al. 2019) [2] against antigenic challenges. Interleukin-1 is a key cytokine that produces by many porcine cells such as macrophages and intestinal epithelial cells (Standyk, 2002) [3]. It is well known to influence thymocyte proliferation and B-cell growth and differentiation. It also needed for the production of, interleukin-2, interleukin-6 and chemokine attractions for neutrophils and interleukin-8 (Armstrong et al., 2004). IL-1 not only response during infection but it also affected by appetite, sleep and body temperature (Allen et al. 2005 and Dinarello et al. 2012) [4,5] so it can be used for monitoring the status of animal welfare and health. Increased proinflammatory cytokines generally have also a negative influence on growth and well-being (Stahly et al.1996) [6]. TNF-α is usually produced simultaneously with interleukins and potentializing pro-inflammatory vascular and cellular alterations (Meyer et al. 2003) [7]. Therefore, TNF-α and IL-1 are usually produced together as a prompt response to bacterial infections. Together they both modulate pro-inflammatory signals by coordinating vascular and cellular changes in the immune system, activating many of the components present in the innate immunity, increasing the expression of chemokine and adhesion molecules, critical for the recruitment of neutrophils from the blood, initiating the immune first lineage prompt response in the host (Bazzoni et al.1996, Dinarello et al. 1997 and Mehrad et al. 1999) [8-10]. As inflammation progresses, leukocyte, lymphocyte, and other cells are activated and recruited at inflamed site by a signalling network. Haematological biomarker are key indicators for health and disease surveillance in all animals as well as swine (Mbanasor et al. 2003). Subnormal inflammatory chain reaction in the body generates more free radical which responsible for further adverse effects on health, production, and body weight gain. The importance of pro-inflammatory cytokines (TNF-α and IL-1) and routine a haematological biomarker has kept in mind, study was designed to evaluate the selected parameters, it might be helpful for surveillance of swine health and wellbeing.

Six (6) crossbreed pigs (Yorkshire × Swedish Landrace) were selected with a similar age group (3 years), unisex (male), and 140 kg average body wt for the evaluation of and pro-inflammatory cytokines and haematological indicators values. All the selected pigshad healthy body conditions, normal skin colour, and visually normal physiological activity (Figure 1).

The pig was restrained and bled through the caudal vena cava as described by (Carle and Dewhirst, 1942) into a blood collection tube containing ethylene-diamine tetra-acetic acid (EDTA) as an anticoagulant for hematological study and for serum study blood was collected in plain blood collection viol.

The blood samples were analyzed for total erythrocyte count (TEC), haemoglobin concentration (Hb), packed cell volume (PCV), and total and differential leukocytes count. The PCV was determined using the microhaematocrit method while the RBC and leukocyte counts were determined using the improved Neubauer counting chamber (Jain, 1986) [11]. Smears for differential leukocyte counts were stained by the Leishman technique, and the different cells of leukocyte series were enumerated by the longitudinal counting method. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and meancorpuscular haemoglobin concentration (MCHC) were calculated from the PCV, Hb, and RBC count.

Serum concentrations of interleukin-1 and tumor necrosis factor -α were determined with commercially available ELISA kits for detection of porcine cytokines by using the described protocol by (Chongqing Biopes, Co, Ltd. China). Cytokines assay (TNF and IL-1), were evaluated in each well of ELISA plate at last step addition of kit component 7 and finally mixing thoroughly, it produced immediately color changes into yellow that showed (Figure 2). After the appearance of yellow color noted the ELISA plate readingand calculated the average mean values of 6 samples.

Descriptive analysis is presented as individual value, group mean and standard error. Pearson’s correlation coefficient was calculated for the relationship between all qualitative variables by using Excel Micro software.

Mean and individual serum values of cytokines of adult porcine are presented in Table 1 and Graph A and B. The mean cytokines values (n=6) of TNF-α was 242.33± 10.23 ng/l and IL-1 was12.16±1.16. The mean values of cytokines were statistically non-significant with individual value as well as reference values. However, the highest value of TNF-α and IL-1 was measured to be 290.0 ng/l and 19.0 ng/l while the lowest was 230.0 ng/l and 9 ng/l respectively.

Statistical analysis of haematological indicators values are presented in Table 2 and Graph C and it showed that, mean red blood cell count was 6.43±0.14 10 6/μL, mean haemoglobin concentration was 12.33±0.33g/dl and mean PCV was 36.5±1.1%. RBC indices were also ranges with reference values in growing porcine. Mean count of WBC was showed moderate increment under reference range. The percentage of neutrophils was found to be lower but the percentage of lymphocyte and eosinophils were observed comparatively higher than references values of other domestic animals respectively.

Correlation level of pro-inflammatory cytokines and routine haematological biomarker are present in Table 3 and Graph D. Correlation coefficient values showed that, positive correlation between IL-1 and other three variables TNF-α, WBC and neutrophils except lymphocytes that was showed very poor correlation with IL-1 as well as negative correlation with TNF-α (-0.21), WBC (-0.02) and neutrophils (-0.71).

Pro-inflammatory cytokines and routine haematological parameters are reliable indicators for the assessment of animal health and wellbeing (Wolmarans et al., 2011) [12]. Cytokines bioassay of TNF-α and IL-1 revealed that the concentration of both the cytokines was similar to findings of previous studies. (Beisel et al.,1991 and Stahly et al., 1996) [6,13]. Serum concentration of proinflammatory cytokines are directly influenced by what the animal exposed to be, it viruses, intracellular or extracellular bacteria, myco-toxin or non-infectious damage (Nordgreen et al., 2020) [14]. Unhygienic environments are typically dusty and exposure to dust triggers a cytokine response (Sahlander et al., 2012) [15]. Gaseous component of air like ammonia causes the releases of cytokines by alveolar macrophages and neutrophils (Murata et al., 1999) [16]. Psychological stress one of the most important inducing factors of proinflammatory cytokine production in humans as well as rodent (Maes et al., 1998) [17]. In the pig, psychological stress might affect the immune response (Manciocco et al., 2011) [18]. Protein deficient diet particularly tryptophan under low sanitary the condition can influence the cytokine production in pig (Nordgreen et al., 2020) [14]. Release of cytokines activated the cellular and humoral components of the immune system such as phagocytes and antibodies. The active immune system of the body is singling the rearrangement dynamics of perivascular blood components.

In the statistical analysis haematological biomarker of health status showed that non-significant differences between lower and higher mean values of total erythrocyte count, haemoglobin, HCT, MCH, MCHC, and MCV. It means, the health status of swine could be varying in terms of minerals, vitamins levels oxygen-carrying capacity of the blood, and hydration status of the body. A significant difference was found between lower and higher mean values of total leukocyte counts. It might be due to the different immune status of individuals. Acute tissue damage or bacterial invasion in the body generates a wide variety of signals for the early recruitment of neutrophils (De et al., 2016) [19] lead to excessive migration of neutrophils towards the peripheral circulation. Lymphocytes usually respond to foreign antigens only if the innate immune system is first activated (Alberts et al., 2002) [20]. Clinical and subclinical viral infection enhances lymphocyte proliferation, expressed in the peripheral circulation. Monocytes are recruited to infected tissues and mediate direct antimicrobial activity at this site (Serbina et al., 2008) [21]. Basophils are the least abundant leucocytes primarily found in the circulation, but rapidly expand in the bone marrow in response to inflammatory signals and are mobilized to the blood, spleen, lung, and liver (Min, et al., 2012) [22]. Eosinophils have classically been associated with host defense against parasitic infections, particularly caused by helminths (Taenia solium) in swine (Ramirez et al., 2018) [23].

Correlation coefficient, statistical measured for analyzed the strength of the relationship between selected parameters (Table 3 and Graph D) [24]. IL-1 were more positively correlated with TNF-α (r=0.87), WBC (r=0.66), Neutrophils (r=0.63), and non-significantly positive with Lymphocytes(r=0.02).It indicates that initial active immune responses positively triggered the TNF-α, WBC, and Neutrophils' action. TNF-α were also showed significantly positive correlation with IL-1 (r=0.87), WBC (r=0.48), Neutrophils (r=0.73), and non-significantly negative with Lymphocytes(r=-0.21). It means that initial active immune responses positively triggered the TNF-α, WBC, and Neutrophils' action but negatively with lymphocytes because activation is a delayed process.WBC positively correlated with all the variables except lymphocyte. The relationship between Neutrophils and lymphocytes were showed significantly negative. The relationship of lymphocyte with other variables showed negative except IL-1.

The study concluded that each value of the immunological parameter and haematological biomarker was found to be within normal reference range while at individual-level some variation showed because of seasonal and nutritional variation can be considerable. Particularly, immunological TNF-α is more closely associated with proinflammatory effects and IL-1 was originally named "lymphocyte activating factor" because it stimulates lymphocyte proliferation in the presence of suboptimal doses of mutagen. On the basis of the above finding, it can be concluded that pro-inflammatory cytokines and haematological biomarker is good and reliable parameters for assessment of health status and welfare in the pig industries of India.