IntroductionTop

Escalating mastitis cases courtesy mainly the mismanagement and emergence of antibiotic-resistant bacterial strains are causing great economic breakdown across the world (Aslantaş & Demir, 2016). Mastitis, an inflammatory disease of the mammary gland, is caused by a wide variety of pathogenic microorganisms. Among them, Staphylococcus aureus and Escherichia coli are the most common (Bannerman, 2009). With multiple aetiological agents, mastitis problems remain vague via strategic use of the effective vaccine (Detilleux et al., 1994). Apart from managemental care, selection of disease resistant animals into the herds is a promising approach for resolving mastitis. Genetic selection of cattle populations in Scandinavian countries has been performed on the basis of udder health and recorded clinical mastitis case (Heringstad et al., 2007). The inclusion for sire evaluation in these breeding program­mes has resulted in a lower trend of clinical mastitis cases in these countries (Sender et al., 2013).

The innate immune system of an animal tries to combat a broad variety of pathogens and is based on a limited repertoire of germline-encoded receptors called pattern recognition receptors (PRRs) of immunocytes. PRRs of immunocytes sense specific Pathogen Associated Molecular Patterns (PAMPs) of pathogens and lead to the production of cytokines which in turn bridge innate and adaptive immune responses (Mogensen, 2009). Lipopolysaccharide (LPS), the outer component of gram-negative bacteria such as E. coli, binds with LPS binding protein that in turn binds with CD14/TLR4/MD2 receptor complex and eventually induces cytokine release from various kinds of cells (Gerold et al., 2007; Lu et al., 2008). Through its involvement in different pathways, LPS has been reported to induce a pro-inflammatory response in different mastitis-models (Xiao et al., 2015). White blood cells of the vascular system especially lymphocytes, monocytes and macrophages effectively regulate the inflammation process by acting as the first line of immune defence against invading pathogens (Mogensen, 2009). These cells have the ability to produce inflammatory mediators like the cytokines and chemokines that act by attracting other immune cells to the site of inflammation. Lymphocytes that invade the mammary gland during infection are present in its secretions and participate in an active role by providing protection against important mastitis causing organisms (Park et al., 2004).

Animal hosts, with diverse genetic makeup, enco­unter numerous pathogens with varying virulence. The genetic make-up, physiological state of the host and the traits of the etiological agents govern about the disease tolerance and/or susceptibility in the host (Zanella, 2016). Disease resistance is a threshold trait which follows polygenic inheritance but varies phenotypically in a discontinuous manner i.e., either normal or diseased (Falconer & Mackay, 1996). Breed-wise and species-wise differences in susceptibility pattern towards mastitis have been well documented (Rupp & Boichard, 2003). Buffaloes in comparison to crossbred cattle and Jersey cows in comparison to Holstein cows have been reported to have a lower risk and consequently the prevalence of mastitis (Iqbal & Siddique, 1999; Kelm et al., 2001; Youngerman et al., 2004; Biffa et al., 2005; Berry et al., 2007; Jingar et al., 2014a,b). Jingar et al. (2014b) reported the incidence of mastitis to increase significantly with increase in parity number in cattle species, but not in buffalo.

Dissimilarity in the character of the inflammatory response also influences the potency of the host to control intramammary invasions (Bannerman et al., 2008a). However, the immune response in the form of production of cytokines as a result of encountering mastitis pathogens was found to be similar (Bannerman et al., 2008a,b). Genetic basis of resistance to infectious diseases in animals depends on additive genetic variation that exists among animals and gives a differential response to various infectious challenges (Yañez et al., 2014). Therefore, it is imperative to study the genes responsible for disease resistance to mastitis associated pathogen and other pathogens. The aim of this study was to identify, evaluate and compare early phase immune response associated genes in peripheral blood mononuclear cells (PBMCs) isolated from blood of crossbred cattle, Tharparkar cattle and Murrah buffalo in response to E. coli’s LPS stimulation. It was done through the evaluation of relative expression of the transcripts by real-time PCR in order to explore out any breed-wise and species-wise difference(s) in resistance pattern towards mastitis.

Material and methodsTop

Selection of animals for blood collection

All animals used in the experimentation study belonged to the Cattle and Buffalo Farm, ICAR-IVRI (Izatnagar, Bareilly, UP, India), and the animal experimentation was performed in strict compliance with the rules and regulations set by the Institutional Animal Ethics Committee (IAEC). Apparently healthy lactating crossbred Vrindavani cattle (rotational cross having 50–75% exotic inheritance viz. Holstein-Friesian, Jersey and Brown Swiss; with the rest coming from the native indigenous component Hariana cattle breed), Tharparkar cattle and Murrah buffaloes were taken for the study. The animals selected were mainly in their 3rd or 4th parity with no clinical history of mastitis and screening of milk samples was performed for different mastitis specific tests.

Procedure for initial screening of samples

The milk samples of clinical mastitis free animals were screened for California mastitis test (CMT) score and milk somatic cell count (SCC) to rule out any presence of subclinical mastitis. Milk samples of 60 animals (45 Crossbreds cattle, 15 Tharparkar cattle and 15 Murrah buffaloes) were screened by CMT and SCC. Among these, six animals from each group (crossbred, Tharparkar and Murrah) were selected as experimental animals, whose SCC were less than 200,000 cells/mL of milk and whose milk samples showed a CMT score of either 0 or 1. Blood (4 mL) from the six selected experimental animals of each group was collected for PBMCs isolation by density gradient centrifugation method (Histopaque-1.083 g/mL, Sigma, Poole, Dorset, UK).

PBMCs culture and stimulation with LPS

The recovery and viability of the isolated PBMCs were checked by Countess™ Automated Cell Counter (Invitrogen, USA). Viable blood mononuclear cells obtained from each animal were plated at a density of 1×106 cells/mL (3×106 cells/well) into two rows of polystyrene culture plate wells. One row of culture plate wells were stimulated with E. coli outer membrane antigen LPS (Sigma-Aldrich, Saint Louis, USA) at a dose rate of 1 µg/mL of culture media with stimulation time of 6 h and another row was kept un-stimulated as a control for this study. The culture plates were kept at 37ºC with 5% CO2 levels. In vitro stimulation of bovine PBMCs was done with LPS of E. coli at a pre-determined standardized dose as described by Doherty et al. (2013). The time period of 6 h was considered optimum for our study because most of the inflammatory cytokines after reaching an expression peak at about 4 to 6 h were either found to have no change or showed a decrease in the level of cytokines after 12 or 24 h of challenge (Strandberg et al., 2005). The supernatants obtained after centrifugation and separation of PBMCs were pooled for each animal and stored at -80ºC to be used for further estimation of IFN-γ protein by ELISA. All the supernatants were used within 48 h for ELISA protocol.

Total RNA extraction and qRT-PCR analysis

The differential mRNA expression levels of IL-10, IFN-γ, IL-1β, IL-12β, CCL-5 genes in stimulated and unstimulated PBMCs were analysed by real-time PCR using Fast SYBR® Green dye (Applied Biosystems, Warrington, UK). The real-time PCR system (Step one plus, Applied Biosystems, Warrington, UK) was used with the already published and self-designed gene-specific primers mentioned in Table 1. RNA isolation from stimulated and unstimulated samples of PBMCs was carried out using RNeasy Plus Mini Kit (Qiagen) and the manufacturer’s instructions. The extracted RNA was quantitated in NanoDrop ND 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purity of RNA was evaluated by measuring the standard absorbance ratio for RNA (A260/A280). Synthesis of first strand cDNA was done using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, USA) following the manufacturer’s instructions. Final Concentration of cDNA was adjusted to the level of 5 ng/µL. The thermal cycling program of the qPCR included one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. A dissociation step was also included to confirm the specificity of amplification. Six biological samples with three technical replicates of each biological sample were used in the present study. A negative template control was also included in the qRT-PCR reaction.

The primers for the selected genes were either chosen from the reference paper(s) or designed with the help of Integrated DNA technologies (IDT) software Primer­Quest Tool (Coralville, IA, USA). For designed sets of primers, reaction efficiencies were calculated using a two-fold serial dilution of pooled cDNA. A standard curve was constructed by plotting average cycle thresholds (CTS) against log10 (cDNA concentration/100). The line obtained from the slope (m) was then utilized to calculate the efficiencies of primers using the formula 10(−1/m)−1. Primers with efficiencies between 0.8 and 1.1 were considered satisfactory and were included in the study (Table 1). β-Actin was used as a reference gene for normalization of expression data (Doherty et al., 2013). The production of the melt curve as a result of the dissociation was observed for the presence of a single peak that shall represent a single PCR product for each gene amplified.

Estimation of IFN-γ by ELISA

IFN-γ levels were estimated in the supernatants obtai­ned after centrifugation of the stimulated and unstimulated PBMCs via ELISA technique. The supernatant from six samples of each group of animals was collected. The samples were pooled together in a set of two for carrying out the ELISA protocol. Each of the pooled samples was tested in duplicate and IFN-γ was detected by the standard protocol (Cusabio, Hubei Province, China). The dilutions to which the sera were tested were up to 1:50. Standard and sample (100 µL) were added per well in 96-well plates, which were then incubated for 2 h at 37oC. Biotin antibody (100 µL) was added to each well and again incubated for 1 h. After washing three times, 100 µL of horseradish peroxidase (HRP) avidin was added to each well and incubated for 1 h. Plates were washed again five times following the standard procedure and then 90 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well and incubated for 30 min and then 50 µL of stop solution was added. The optical density of each well was then determined within 5 min, using a microplate reader set at 450 nm.

Statistical analysis

qRT-PCR data were converted to log2 fold changes relative to control unstimulated samples using the 2 −ΔΔCT method. Comparative cycle threshold (CT) method (Schmittgen & Livak, 2008) was used for analysis of the generated data. The non-parametric Mann-Whitney U Test was performed to determine significant differences between ΔCTS of the analyzed groups. Statistical ana­lysis of the obtained ELISA data was done by using Tukey’s Honest Significant Difference test by using SAS 9.2 version (SAS Inst., Cary, NC, USA).

ResultsTop

Cytokine mRNA expression of PBMCs against LPS challenge

PBMCs isolated from blood of the three groups (crossbred, Tharparkar and Murrah buffaloes) were stimulated with pre-determined standardized LPS dose of E. coli as a TLR-4 agonist in order to monitor the differential mRNA expression of CCL5, IL-1β, IL-12β, IFN-γ and IL-10 genes with respect to unstimulated PBMCs. qRT-PCR technique was employed to esta­blish any species-wise and breed-wise differential mode and intensity of immune response against a similar dose of antigen from the similar pathogen. Comparative cytokine profile analysis of in vitro stimulated PBMCs of the three groups with same pre-standardized LPS dose were made. As shown in Fig. 1, the expression level of CCL5 gene was significantly up-regulated in stimulated PBMCs of crossbred (p<0.01) and Tharparkar (p<0.01) and was unaffected in PBMCs of Murrah when compared to un-stimulated control. The expression level of IL-1β gene was significantly (p<0.01) up-regulated, down-regulated and unchan­ged in stimulated PBMCs as compared to unstimu­lated PBMCs of crossbred cattle, Murrah buffaloes and Tharparkar cattle, respectively as shown in Fig. 1.

Figure 1. Relative fold change expression of genes in Murrah (BL), Tharparkar (TL), and crossbred (VL), respectively. One way ANOVA was performed to determine the significant diFferences between ΔCTS of BL, TL, and VL for CCL-5, IL-1β, IL-10, IL-12β and IFN-γ gene respectively. *, **, *** indicate significance at p<0.05, p<0.01 and p<0.001, respectively. The non-parametric Mann-Whitney U Test was performed to determine the significant differences between ΔCTS of the challenged groups (Murrah, Tharparkar, and crossbred) with its own control. The figure depicts –0.11, 1.05 (p<0.01) and 2.72 (p<0.01) log2 fold difference of CCL5; –1.75 (p<0.01), 1.08 and 1.55 (p<0.01) log2 fold difference of IL-1β; 0.40, –1 (p<0.01) and 1.06 (p<0.01) log2 fold difference of IL-10; –1.76 (p<0.01), 0.96 (p<0.01) and 4.15 (p<0.01) log2 fold difference of IL-12β and –1.22 (p<0.05), –0.03 and 1.58 (p<0.05) log2 fold difference of IFN-γ mRNA expression respectively of the analysed group with its control.

IL-12β gene showed significant upsurge (p<0.01) in stimulated PBMCs of Tharparkar and crossbred cattle but showed a significant decline (p<0.01) in stimulated PBMCs of Murrah buffaloes with respect to unstimulated control PBMCs (Fig. 1). In the case of IFN-γ, significantly higher (p<0.05) expression, and significantly lower (p<0.05) expression and unchanged expression was respectively observed in stimulated PBMCs of crossbred cattle, Murrah buffaloes and Tharparkar cattle as shown in Fig. 1. The expression level of IL-10 was significantly higher (p<0.01), lower (p<0.01) but unchanged in stimulated PBMCs of crossbred cattle, Tharparkar cattle and Murrah buffaloes, respectively (Fig. 1).

Comparative analysis of ΔCTS of the CCL5, IL-1β, IL-12β, IFN-γ and IL-10 between crossbred, Tharparkar and Murrah revealed differential regulation of gene expression. Significant differences for CCL5, IL-1β, IL-12β and IFN-γ and non-significant difference for the IL-10 gene were observed between Murrah and crossbred cattle (Fig. 1). Tharparkar samples showed a significant difference for only IL-12β and IL-10 genes when compared with crossbred ones (Fig. 1). On the other hand, the comparison between Murrah and Tharparkar showed significant differences for IL-1β and IL-12β genes only (Fig. 1).

IFN-γ detection in PBMCs culture supernatant – ELISA approach

Tukey’s HSD test revealed no significant difference in the level of IFN-γ in stimulated (LPS) and unstimu­lated groups of Tharparkar and Murrah PBMCs super­natant but showed a significant difference (p<0.01) in the stimulated group when compared with the unstimu­lated group of crossbred cattle (Fig. 2).

Figure 2. Average concentration of IFN-γ in stimulated (LPS) and unstimulated groups of crossbred, Tharparkar and Murrah cell culture supernatants. ** indicates significance at p<0.01.

DiscussionTop

Mastitis resistant pattern of the host depends upon infectious dose, route of infection and virulence of pathogen strain, immune status of the host, mode and intensity of the immune response of host against encountering pathogen, etc. Identification of differentially expressed genes in response to a pathogen may be useful to identify important markers which in turn can serve as an important tool for selection of animals showing more tolerance towards this complex polygenic trait (Fonseca et al., 2009). For the first time, this study was undertaken for Tharparkar cattle and Murrah buffaloes in order to identify differentially expressed genes in response to E. coli infection.

E. coli-LPS, a major outer membrane antigen, binds with CD14/TLR4/MD2 receptor complex and induces cytokine release in various types of cells (Gerold et al., 2007; Lu et al., 2008). IFN-γ, a pro-inflammatory cytokine secreted by immune cells acts as an important link between innate and adaptive immunity and is very important against the intracellular pathogens (Bannerman, 2009). Its concentration is known to increase in sera during coliform mastitis (Hisaeda et al., 2001). Un-altered and significantly down-regulated INF-γ transcripts levels in stimulated PBMCs of Tharparkar cattle and Murrah buffalo respectively, whereas, significantly higher INF-γ transcript levels in stimulated PBMCs of crossbred cattle at a similar stimulant dose of E. coli LPS, suggest that immune system of each breed of cattle and also among different species combat encountering pathogens in a different manner. Results of ELISA test of INF-γ done on PBMCs supernatants of the cultures also gave indications in support of the real-time PCR results. INF-γ greatly enhances the ability of macrophages and neutrophils to phagocytose and eliminate invading pathogens (Ellis & Beaman, 2004). It also increases the activity of major histocompatibility complex (MHC) class-I molecule and finally promotes cell-mediated immunity by helping cytotoxic T-cell recognition of antigens presented from foreign microbes (Schroder et al., 2004). Higher IFN-γ transcript levels were also detected in milk cells isolated from mammary glands of animals infected with E. coli (Lee et al., 2006).

On the other hand, mRNA expression of IL-12β display27ed a significant increase in both crossbred and Tharparkar which was in concurrence with the result of Lee et al. (2006) but the IL-12β expression in Murrah PBMCs was significantly down-regulated. Expression in crossbred significantly varied with both Tharparkar and Murrah, and there was significant variation between the expression of IL-12β gene among Tharparkar and Murrah too. The results suggest that the immunocytes of all the three groups manifested a non-identical response when stimulated with the same dose of antigen from E. coli. IL-12 produced by antigen-presenting cells such as macrophages after activation of PAMP and Damage Associated Molecular Pattern (DAMP) acts as a strict regulator of Th1 type of immune response and activates Natural Killer (NK) cells to secrete IFN-γ, which in turn further stimulates enhanced secretion of IL-12 (Trinchieri, 2003; Vignali & Kuchroo, 2012; Pérez-Cordón et al., 2014).

IL-1β takes part in several activities such as cell proliferation, cell apoptosis, cell differentiation, leukocytes activation, systemic induction of fever and acute phase protein synthesis (Dinarello, 1996; Barksby et al., 2007). Up-regulated, unchanged and down-regulated expression levels of IL-1β gene in stimulated PBMCs as compared to unstimulated PBMCs of crossbred, Tharparkar and Murrah, respectively in response to a similar dose of E. coli may be reminiscent of differential inflammation regulatory pattern and activation of leucocytes in order to combat E. coli infection. Upsurge IL-1β level in response to LPS in crossbred concur the findings of Strandberg et al. (2005) who also observed increased levels of mRNA expression of IL-1β after 2-4 h of in vitro stimulation of bovine primary mammary epithelial cells by LPS.

CCL5 (Chemokine ligand 5), also known as RANTES, has a role in the inflammatory and immunoregulatory control and has been found to be important for chemotaxis and maintenance of inflammation inside bovine mammary gland (Ezzat Alnakip et al., 2014). CCL5 acts as chemo-attractant for memory T-helper cells, blood monocytes and eosinophils (Song et al., 2015). Upsurge cytokine and chemokines expression level in response to LPS was also in accordance with the finding of Taraktsoglou et al. (2011). Challenge of bovine macrophages with LPS revealed a significant up-regulation of proinflammatory gene CCL5 in PBMCs of crossbred and Tharparkar only. CCL5 gene acts as an important chemokine during clinical mastitis cases and is involved in the recruitment of inflammatory cells and neutrophils to the site of inflammation (Taraktsoglou et al., 2011).

IL-10, a major anti-inflammatory cytokine, is produced in order to limit the excessive release of pro-inflammatory cytokines (IL-1, IFN-γ, IL-12 and TNF-α), chemokines and eicosanoids to suppress Th-1 type response and enhance Th-2 type of response which in turn drive humoral immune response preferentially (Moore et al., 2001; Asadullah et al., 2003; Bannerman, 2009). Up-regulated, down-regulated and un-altered IL-10 expression level in stimulated PBMCs of crossbred, Tharparkar and Murrah, respectively, in spite of similar stimulant dose of E. coli, may suggest species-wise and breed-wise different degree of the immune response against pathogen invasion. The result may be suggestive of the intensity of immune response against pathogen and the somewhat conclusive idea of mastitis resistant pattern which may vary according to breed and species. Animals with intramammary E. coli infection revealed an increased concentration of IL-10 in milk (Bannerman et al., 2004).

Several studies suggest the role of a proper balance between different pro-inflammatory cytokines and anti-inflammatory cytokines in combating mastitis caused by the invasion of a variety of bacterial organisms (Stelwagen et al., 2009). E. coli infection leading to clinical mastitis causes high up-regulation of pro-inflammatory cytokines which may cause tissue damage or even death of the animals by inducing severe clinical mastitis and fever. A higher level of pro-inflammatory cytokines can be counterbalanced by an increase in the level of anti-inflammatory cytokine-like IL-10, so administration of such anti-inflammatory cytokines can be beneficial to counteract the effect of pro-inflammatory cytokines (Bannerman, 2009). It has been documented that cattle shows substantial genetic variation for disease resistance including mastitis (Morris, 2007; Sender et al., 2013; Litwińczuk et al., 2015). Breed-wise differences in mastitis occurrence have also been reported by Nóbrega & Langoni (2011). Crossbred stimulated PBMCs culture showed higher expression levels of CCL5, IL-1β, IL-12β, IFN-γ and IL-10 with respect to unstimulated culture. Tharparkar stimulated PBMCs showed a significant upsurge in CCL5 and IL-12β expression level only but lower expression level of these genes in comparison to crossbred, along with downturn of IL-10 expression and non-significant alteration in IL-1β and IFN-γ expression level as compared with unstimulated PBMCs. The results give an indication that similar doses of the lipopolysaccharide were not able to induce a similar increase in response of pro-inflammatory cytokines in Tharparkar in comparison to crossbred and the expression of anti-inflammatory cytokine thus showed a downturn in Tharparkar as it was not needed to control the over-expression of pro-inflammatory cytokines. The unchanged expression of IL-1β gene in Tharparkar PBMCs also gave a sign of positive immune response as the excessive release of IL-1β has the ability to induce vascular leakage, shock and multi-organ failure. This may also indicate a more balanced immune response of Tharparkar in comparison to crossbred. While Murrah stimulated PBMCs exhibited significant recession in mRNA level of IL-1β, IL-12β and IFN-γ, but non-significant alteration in CCL5 and IL-10 mRNA level as compared with unstimulated PBMCs. Our findings reaffirm the previous findings of Dewangan et al. (2015) and Panigrahi et al. (2016), which also suggested differential expression gene pattern in immune-related genes of PBMCs of crossbred, Tharparkar cattle and Murrah buffalo after stimulation with similar dose of T. annulata sporozoites, which may account for difference in disease resistance pattern to T. annulata infection among different species and breed. Bannerman et al. (2008a,b) working on a comparison of Holstein and Jersey innate immune response by intramammary infection of E. coli and S. aureus, were not able to find any differences between these two breeds and reported a highly conserved innate immune response of these breeds. However, Sulabh et al. (2018) reported the differential response of mRNA transcript of cytokines between stimulated PBMCs culture of crossbred, Tharparkar cattle and Murrah buffalo when they were stimulated with the same dose of peptidoglycan and lipoteichoic acid obtained from S. aureus. Differences in innate immune responses against various bacterial infections have also been reported by Fonseca et al. (2009) and Gibson et al. (2016).

Our results further showed the contrasting differences in the expression level of IFN-γ, IL-1β and IL-12β genes of Murrah in comparison to crossbred. This may suggest that PBMCs of Murrah may require a higher dose of antigen challenge. It also gave an indication that immunocytes of Murrah may be refractory to lower dose of antigen challenge and may require a higher number of E. coli bacteria to elicit an inflammatory response or to induce disease in these animals in comparison to crossbred; this may be one of the prime reasons for the lower incidence of mastitis in buffaloes. Yu et al. (2010) used various concentrations (1 ng/mL to 1 µg/mL for polymorphonuclear leucocytes for 6 h) in which the results highlighted an increased response of gene expression with increasing dose. However, for mRNA expression of GCP-2 and IL-8 they also used an additional dose rate of 10 µg/mL (for 24 h), which showed an increase in the expression of the concerned genes. However, proteomic studies, post-transcriptional modification, epigenetic regulation and inclusion of a large number of animals in the study are also required to confirm conclusive idea for genetic markers of mastitis resistant pattern of host apart from monitoring of relative immune-related gene expression level with stimulating LPS dose of E. coli.

The results of this study suggest that the immunocytes of the three groups manifested non-identical response when stimulated with the same dose of antigen from E. coli. It points towards the presence of differential immune response among different breeds/species, which have evolved as a result of evolutionary adaptation to a particular environment or specific man-made selection(s) that was responsible for the creation of different species/breeds of animals. This possibly indicates the role of the immune system in causing the differential outcome of a disease in response to the pathogen invasion.

AcknowledgementsTop

The authors highly acknowledge the help and support rendered by the Director, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India, for providing necessary funds and important facilities to carry out this research.