Introduction

Foot-and-mouth disease (FMD) is one of the most contagious and highly infectious viral diseases of cloven-hoofed animals with a devastating impact on the livestock economy. Clinical signs of FMD are high fever, anorexia, hypersalivation, blisters on the foot and mouth, and lameness. The disease is rarely fatal in adult animals but often leads to high morbidity and decreased productivity that persists even after recovery from clinical infection (Chowdhury et al. 1993; Ferrari et al. 2014). In India, FMD is endemic and poses a constant risk to the livestock sector.

Heat shock proteins (HSPs) are a group of evolutionary conserved proteins present nearly in all cells in all life forms. In the last few decades, HSPs have received much attention for their intracellular chaperone activity during thermal shock. In addition, the involvement of HSPs in other stressful situations including bacterial and viral infections has also been confirmed (Ungar-Waron et al. 1996; Njemini et al. 2003). Although HSPs are primarily intracellular, they can be released into the extracellular milieu during stress (Vega et al. 2008). Within the family of HSPs, HSP70 is the most abundant and well-characterized protein chaperone (Hunt and Morimoto 1985). In farm animals, several studies have documented HSP70 levels in the blood (Kristensen et al. 2004) and other biological fluids (Lamy et al. 2017), and peripheral HSP70 levels have been reported to be elevated in cattle during heat stress (Mishra et al. 2011; Gaughan et al. 2013). Heat shock response following infection is one of the intriguing but less studied aspects. Study in human subjects has proposed extracellular HSP70 level as a criterion to distinguish between patients with infection and healthy individuals (Njemini et al. 2003). Previously, over-expression of HSP70 has been reported in FMD virus infected heart tissues of lambs (Gulbahar et al. 2011). In recent studies, HSP70 when used as an adjuvant in novel FMD vaccine formulations boosted immune response in mice and pigs (Sedeh et al. 2014; Lee et al. 2020). At this juncture, understanding impact of FMD viral infection on HSP70 response at the whole organism level may be insightful and informative.

Infection burdens are often accompanied by endocrine alterations and inflammatory responses. Cortisol is well-recognized as the major stress hormone in mammals (Martin et al. 2011). Many stressors including disease processes can activate the hypothalamic–pituitary–adrenal (HPA) axis. Activation of the HPA axis can mediate metabolic and immune system effects (Bailey et al. 2003). The published literature on the impact of FMD on serum cortisol levels is limited and the results are contradictory (Ghanem and Abdel-Hamid 2010; Barkakati et al. 2016). It has been further postulated that FMD viral infection interferes with the hypothalamic-pituitary-thyroid axis (Artz et al. 2011). Few researchers have investigated thyroid functionality during experimental FMD (Maddur et al. 2011; Saravanan et al. 2020); however, serum T3/T4 levels following natural FMD infection in cattle are still largely unknown.

Acute-phase proteins (APPs) are hepatocyte derived proteins whose blood concentrations change in response to infection, inflammation, or stress (Murata et al. 2004). APPs are used in veterinary medicine as additional biomarkers for animal disease diagnoses (Godson et al. 1996). Haptoglobin and serum amyloid A are the two major APPs (Horadagoda et al. 1999) in cattle. The degree of change in APPs was related to the clinical severity of FMD (Merhan et al. 2017). However, the association between acute-phase response protein and hormonal changes during FMD viral infection is poorly understood. Moreover, whether these changes are associated with circulating HSP70 concentrations is not known. Therefore, the present study aimed to investigate serum levels of HSP70 and its correlation if any with serum cortisol, thyroid hormones, and acute-phase protein concentration in FMD infected and clinically healthy cattle. To the best of our knowledge, changes in serum concentrations of HSP70 have not been explored under pathological conditions of bovines.

Materials and methods

After the FMD outbreak in natural conditions in an organized dairy (Holstein Friesian × Zebu) cattle herd in India, blood samples were collected from diseased (n = 40) and apparently healthy cattle (n = 30). The serum was separated after centrifugation at 3000 rpm for 10 min. Serological confirmation of FMD infection was done by in-house 3AB3 based indirect DIVA ELISA (Mohapatra et al. 2011). Concentrations of HSP70 (MBS7606199), cortisol (MBS028594), thyroxine (T4) (MBS017877), tri-iodothyronine (T3) (MBS014516), haptoglobin (MBS033026), and serum amyloid A (MBS041375) were estimated by commercial ELISA (MyBiosurce, SanDiego, California, USA) kits. Details regarding intra-assay and inter-assay coefficients of variation are presented in Supplementary Table S1. An unpaired t test was applied to compare the group means for various parameters. Pearson’s correlation coefficient was used to find the association of HSP70 level with other serum parameters for each group. Scatter plot with simple linear regression was used to statistically describe the related variables. A p value < 0.05 was considered statistically significant. Graphpad prism software version 5 (GraphPad Software, Inc., San Diego, CA) was used for the statistical analysis of the data.

Results and discussion

The mean (± SE) concentrations of the blood parameters are presented in Table 1. In the present study, HSP70 was detectable in the sera of both group of animals and values are comparable with other observations for extracellular HSP70 concentration in cattle (Gaughan et al. 2013; Lamy et al. 2017). Serum levels of HSP70 were significantly higher (p < 0.01) in FMD infected animals than apparently healthy ones. Rise in circulating HSP70 possibly reflecting induced intracellular production of this stress protein. However, the origin of free HSP70 in circulation is still not defined and implies multiple cellular provenances (Mambula and Calderwood 2006). There are also suggestions that fever in the face of acute infection can induce HSP70 in intracellular and extracellular space (Gupta et al. 2013). Increased circulating HSP70 during FMD progression presumably stimulate the host defense mechanism; however, further studies are warranted to establish whether elevated extracellular HSP70 level play pro-viral or pro-host function in FMD.

Table 1 Comparison of blood serum parameters in healthy and FMD infected cattle
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Stress imposed by any disease further impacts animal health and performance. Serum cortisol levels in FMD infected cattle were significantly higher (p < 0.05) in relation to healthy animals. High magnitude of systemic cortisol refers to disrupted homeostasis in FMD and this may be an outcome of increased stress on the animals due to infection. On the other hand, we observed a marginally higher serum T4 concentration in infected animals but the overall changes were not statistically significant (p > 0.05). Serum T3 levels also did not vary (p > 0.05) between healthy and infected group. Madur et al. (2011) reported a similar thyroid response in experimentally infected cattle while Saravanan et al. (2020) showed that T3 and T4 concentrations returned to pre-infection level after transient hyperthyroidism following the FMDV challenge in calves. The subtle variation in both the observations may be due to the difference in age of the experimental animals. Our data indicate that FMD virus infection does not affect thyroid gland activity.

In this study, we observed large interanimal variation in serum haptoglobin concentrations. Nevertheless, a markedly higher (p < 0.001) haptoglobin level was found in infected cattle sera compared to the healthy group. Serum amyloid A concentrations also increased significantly (p < 0.05) in the affected animals. Earlier, in an FMD virus challenge experiment, Stenfeldt et al. (2011) noticed that the rise in haptoglobin and serum amyloid A concentration coincided with the onset of clinical disease and the appearance of viremia. From our observations, haptoglobin is suggested as a more sensitive acute-phase reactant than serum amyloid A after natural FMD infection.

Scatter plot showing positively and significantly correlated parameters in the serum of FMD infected cattle. The solid lines represent the linear regression, r is the Pearson correlation coefficient and p is the significance level.

It is perhaps interesting to note a significant positive correlation of serum HSP70 levels with elevated cortisol (p < 0.05) and haptoglobin levels (p < 0.01) in infected animals (Table 2; Fig. 1 and Fig. 2). Cortisol modulation of tissue HSP70 levels has been earlier demonstrated in fish (LeBlanc et al. 2012). In this study, elevated circulating HSP70 appears to modulate infection related stress and inflammatory response. Previously, corticosterone administration in broiler chickens induced an acute-phase response and HSP 70 expression (Zulkifli et al. 2014) while in human subjects, serum levels of HSP70 were closely related to inflammation status (Njemini et al. 2011). Our results corroborate these findings and substantiate the importance of serum HSP70 level as a danger signal to pathogen attack. No statistical significant, associations (p > 0.05) were observed for HSP70 concentration with other serum parameters in both the groups (Table 2).

Table 2 Correlation of serum HSP70 level with serum cortisol, thyroid hormones, haptoglobin, and serum amyloid A levels in healthy and FMD infected cattle
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Fig. 1
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A significant positive correlation of serum HSP70 levels with elevated cortisol (p < 0.05) in infected animals

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Fig. 2
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A significant positive correlation of serum HSP70 levels with haptoglobin levels (p < 0.01) in infected animals

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In conclusion, serum concentrations of HSP70 increased in FMD infected cattle with a concomitant rise in cortisol, haptoglobin, and serum amyloid A levels compared to healthy cattle. No significant differences were observed for serum thyroid hormone concentrations between the groups. Higher circulating HSP70 levels correlated with cortisol and haptoglobin levels in infected animals indicating that this stress protein may have implications in the disease pathology.