Coccidiosis induces stress hormones in hard ground Barasingha (Rucervus duvaucelii branderi)

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Banerji, K. P. Singh, Nidhi Rajput, Amol Rokde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7252608/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Barasingha kwon as hard ground swamp deer ( Rucervus duvaucelii branderi ) found exclusively in Kanha Tiger Reserve of Madhya Pradesh while small herd where shifted to Satpura and Bandhavgarh tiger reserves under ex-situ conservation plan. Barasingha enlisted in threatened species of Schedule-I and only three sub-species existing in India. During health monitoring and disease diagnosis schedule, physical body score evaluation and disease diagnostics of free ranging barasingha were performed using non invasive techniques. Freshly voided fecal pallets of barasingha were used for two way analysis (i) coprodiagnostic analysis to screen the gastrointestinal parasitic occurrence (ii) assessment of faecal cortisol metabolites using ELISA diagnostic kit (DetecX, Arbor assay, Ann Arbor, Michigan, USA). The extracted cortisol aliquots were analyzed and obtained mean fecal cortisol metabolites (FCM) level 263.335 ± 43.050 pg/ml and 290.054 ± 40.147pg/ml in Barasingha of Kanha and Satpura tiger reserve respectively. Significant positive correlation was observed between FCM concentration with positive fecal samples of Eimeria spp in barasingha of both the parks at p < 0.01 level (2- tailed). Furthermore, significant positive correlation was also observed between FCM level and poor body condition score in Barasingha at p < 0.01 level (2- tailed). The study instigate poor body condition has strong correlation with coccidiosis that increases corticosteroids in barasingha affecting foraging behaviour. Stress evaluation Faecal Cortisol Barasingha Body condition score Kanha Satpura ELISA INTRODUCTION Barasingha known for their gorgeous look and attractive antlers with shy nature amongst deer family called hard ground swam deer enlisted in schedule –I of Wild life (Protection) Act 1972 amended in 2022. In India, distributed sub species of barasingha ( Rucervus duvaucelii branderi ) exclusively found in Kanha tiger reserve of Madhya Pradesh. However, another sub-species of barasingha ( Rucervus duvaucelii ranjitsinghi ) located in the plains of Brahmaputra River in Assam and North East India and sub species of barasingha ( Rucervus duvaucelii duvaucelii) found in the fertile plains of Uttar Pradesh including Nepal (Ahmad. 2007; Bajpai 2015 ). Simplification of ecosystem and urbanization of human colonies beside protected forest areas are major threat to the wildlife conservation enhancing unrest and stressful situations in free ranging wild animals (Wilcox and Colwell, 2005). Depletion in wilderness and encroachment of transitional zones of protected forest area has been building unrest and induce emerging diseases in free ranging wild animals (Whitehouse and Duffus, 2009 ). Deer family of free ranging habitats has pray based importance in the food chain for big cats helps in shaping the grass land meadows (Singh et al. 2019 ). Barasingha serve as important prey for terrestrial mammalian carnivore species while they are agile and sensitive for environmental stressors (Dhami et al. 2023 ). Stress condition stimulates homeostatic, physiological and behavioral responses linked with joint activity of nervous-endocrine system. Anthropogenic interference in the sylvatic cycle sudden change in the nidus, adverse climatic conditions like drought, rain or wind and territorial fighting injuries act as stimulator for stress hormones (Karaer et al. 2023 ). It may induce by many factors and mechanism involving the hypothalamic- pituitary- adrenal (HPA) axis and the sympathetic- adrenal- medullar (SAM) axis. Subsequently the HPA axis is activated leading to release of glucocorticoid hormones while the SAM axis facilitates the release of norepinephrine and epinephrine (Hinds and Sanchen 2022 ). In addition to that the short term release of cortisol is helpful in mobilisation of energy to cope with stressor while chronic release of cortisol leads to muscle wastage, tachycardia and reduced immunity and fertility. In the present study cortisol accession was evaluated in free ranging barasingha ( Rucervus duvaucelii branderi ) those were shifted to Satpura and Bandhavgarh tiger reserves with their close vicinity and similar habitat. These herds were also considered for body score evaluation and correlations with faecal cortisol metabolites along with body conditions and intensity of gastrointestinal parasitic loads that directly affect the foraging behaviour. MATERIALS AND METHODS The herd of barasingha in kanha tiger reserve and Satpura tiger reserves were targeted to collect the biological samples using non invasive techniques: Sample collection The herd of Barasingha were followed early in the morning and observed from a close distance. When the herd moved from their resting place, the freshly laid faecal samples were collected from the pasture. The faecal samples were collected in containers divided into two parts, one part kept without any preservative in cold chain for stress analysis and another part kept in 10% buffered formalin for coprodiagnostic investigation. The samples were brought to school of wildlife forensic and health laboratory, Jabalpur for further processing. Stress evaluation The faecal samples were stored in cold chain and used further dried at 60⁰C for four hours and then powdered finely dispensed in 80% methanol. The mixture vortexed for 30 minutes and centrifuged for 15 minutes at 5000 rpm. The supernatant is collected and stored at -2⁰C according to protocols of Palme et al., ( 2013 ). The supernatant was diluted using assay buffer. The reconstituted diluted samples were run with ELISA assay with following the standard protocol of manufacturer. Coprodiagnostic Investigation The formalin preserved fresh faecal samples were processed using standard techniques as per Soulsby ( 1982 ) including qualitative and quantitative techniques and those samples found higher intensity of parasitic load were further used for EPG (egg/oocyst/ gram) counting. Body condition scoring The animals were photographed with the close vicinity and the body condition was judged based on body evaluation scheme as proposed by Meetei et al. ( 2021 ). The body condition was evaluated based on condition of skin coat, flank ribs, pelvic girdle, vertebral column and lumbar shelf as per the criteria that the animals securing a total score of 0–5 points were adjudged to be in poor; 6–8 points in fair and 9–12 points in good condition. SPSS Statistics software version 30.0 was used for analysis of results. Chi square test was done to study the occurrence of gastrointestinal parasites in Barasingha. Independent sample t-test was done to analyse the difference in stress level in Barasingha in both parks. Correlation analysis was performed to evaluate the effect of different parameters on stress level (Snedecor and Cochran, 1982). RESULTS AND DISCUSSION The studies include body evaluation of barasingha of both tiger reserves along with coprodiagnostics and corticosteroids evaluation focussed on foraging behaviour assessment. According to body condition score (BCS) most of the population of barasingha of either sex were found in good body condition (50.0% animals) at Kanha Tiger Reserve (KTR) in comparison to 38.9% Barasingha in Satpura Tiger Reserve (STR). Poor body condition was revealed in 5.5% barasingha of KTR and 8.3% in STR. However, average body condition encountered in 44.4% barasingha of KTR in comparison to 52.8% barasingha of STR (Table 1 ) Table 1 Body condition score evaluation Tiger Reserve Animal Examined Good (9–12 points) Fair (6–8 points) Poor (0–5 points) Kanha 36 18 (50%) 16 (44.44%) 02 (5.55%) Satpura 36 14 (38.88%) 19 (52.77%) 03 (8.33%) Skin coat = Rough/smooth, Flank and Ribs = Exposed/Muscular, Pelvic girdle = Exposed/Muscular, Vertebral column and Lumbar = Exposed/ Muscular Table 2 Park wise occurrence of gastrointestinal parasites in Barasingha Tiger Reserve Samples Examined Intestinal flukes Strongyles Coccidia Mixed Infection Kanha tiger reserve 36 12 (33%) 4 (11%) 2 (6%) 18 (50%) Satpura tiger reserve 36 9 (25%) 2 (6%) 3 (8%) 22 (61%) X 2 , P 0.605, 0.605 0.727, 0.674 0.215, 1.00 0.900, 0.477 Subsequently, qualitative and quantitative coprodiagnostic investigation has also revealed a mean EPG/OPG 1033.33 ± 185.592 of Coccidia in KTR and 1205.56 ± 192.145 EPG/OPG in Barasingha of STR (Table 2 ). The high rise of stress inducing cortisol was observed in barasingha of KTR and STR those found positive for Coccidiosis ( Eimeria spp) followed by Nematodes (Strongyles) and Intestinal flukes (Amphistomes). Similar results were encountered in wild striped hyaena ( Hyaena hyaena ) population in Southern India with strong correlation of parasitic loads (Ashish, et al. 2023 ) and Bangar, (2019) also emphasized about parasitic load that is responsible for elevation of cortisol metabolites in captive spotted deer ( Axis axis ) and Blackbuck ( Antelope cervicapra ) in India. However, cortisol level also found increased in captive elephants those have high worm load of flukes and Coccidia (Dhairykar, et al . 2022). Furthermore, Mean FCM level was estimated in barasingha as 263.335 ± 43.05 pg/ml in KTR and 290.054 ± 40.157 pg/ml in STR (Table 2 ). Statistical analysis of cortisol concentration in the feacal metabolites showed non- significant difference (p > 0.05) in the mean stress level in barasingha of both tiger reserves. In addition to that significant positive correlation between incidence of poor body condition and FCM concentration through Pearson correlation coefficient of 0.575 was calculated in barasingha of KTR and 0.462 in STR. Both values were found highly significant at 99% confidence intervals. It indicates deterioration of body condition coincides with increase in body FCM level. More or less similar findings encountered in Cervidae species (Pavitt et al. 2016 ; Shah et al. 2022 ). Furthermore, significant positive correlation between poor body condition and FCM concentration in barasingha was observed with positive Pearson correlation coefficient of 0.575 in KTR and 0.462 in STR. Both values were found to be highly significant at 99% confidence interval. It indicates deterioration of body condition coincides with increase in body FCM level. Body condition score of an animal is an indicative of its nutritional intake, parasitic load and its impact on health (Sanchez et al. , 2018). Similar interpretation drawn by George et al. ( 2014 ) who found elevated faecal cortisol levels in European badger ( Melesmeles ) with lower body condition. The authors concluded that poor body condition increases stress in animal and it’s susceptibility to be hunted as prey. Hair cortisol may also be used for metabolic estimation (Potratz et al. 2019 ; Gulzari et al. 2019) found elevated hair cortisol metabolite concentration in deer family which had lower body condition. Humidity is a major contributor to thermal stress in animals and it leads to lower body growth (Perez- Barbaria et al. 2020). However, in the present study more or less similar relative humidity (36–38%) recorded during the entire study period. Notwithstanding, seasonal variations may consider as a tool for stress evaluation. Higher FCM recorded by Corlatti et al. ( 2011 ) in Red deer ( Cervus elaphus ) in Austria during winter compared to summer might be due to low feed intake and lower metabolic rate in summer. Konjevic et al. (2016) also found higher FCM level in adult Chamois ( Rupicapra rupicapra) during summer (186–3271 ng/g) and winter (430–2385 ng/g) compared to spring (129–2896 ng/g). Shah et al. ( 2022 ) also measured higher urinary and faecal cortisol metabolites in captive hog deer ( Axis porcinus) during summer than winter. However, Mostl et al., ( 1999 ) emphasized about rain lead to rise in level of FCM that is probably due to increased microbial intake and consequent rise in gut microbial action to metabolize steroids. According to literature available the present study conducted for the first time to evaluate the cortisol and stress building factors in free ranging barasingha of central India. More affords are needed to decrease the stress in such habitat indicator species for their sustenance in the free ranging habitat. Stress in free ranging animals is affected by a myriad of factors including feed intake, environmental conditions. Coccidian parasite affects the health condition of Barasingha pertaining to foraging behaviour and also contributes to higher FCM level. Non- significant difference noted in the mean cortisol level in the two sites. Poor body condition affects stress level as observed in both the tiger reserves. Declarations No conflicts of Interest : ethical approval and permission from PCCF (Wildlife) Govt. of M.P. was taken for sample collection and procession using non invasive techniques. ACKNOWLEDGEMENTS The authors are thankful to Principal Chief Conservator of Forest (Wildlife) for giving permission to collect the biological sample of barasingha from Kanha and Satpura Tiger Reserve, M.P. under project research work. References Ahmed K. 2007. Ecology and conservation of swamp deer in Terai grasslands of Uttar Pradesh, India. Ph D thesis (Wildlife Science), Aligarh University, India. Ashish K, Arora B, Karne D, Kumar V, Khan A S, Umapathy G, Ramesh T and Kalle R. 2023. Glucocorticoid concentration and parasitic load in a wild striped hyaena ( Hyaena hyaena ) population in Southern India. Theriogenology Wild , 3(1): 100039. Bajpai, S P. 2015. Evaluation of habitat for conserving the hard ground barasingha ( Cervus duvauceli branderi Pocock 1943) in Kanha National Park. Ph.D thesis (Botany), Dr. Harisingh Gour Vishwavidyalaya, Sagar. Bangar N (2019). Faecal cortisol metabolites as an indicator of stress in captive spotted deer ( Axis axis ) and Blackbuck ( Antilope cervicapra ) in India. M.V.Sc. & A.H. thesis (Wildlife Health and Management), University of Nairobi. Corlatti L, Palme R, Frey-Roos F and Hackländer K. 2011. Climatic cues and glucocorticoids in a free-ranging riparian population of red deer ( Cervuselaphus ). Folia Zoologica, 60(1): 176 - 180. Dhairykar M, Singh KP, Jadav, KK and Rajput N. 2020. Comparison of cortisol level in Asian elephants of different tiger reserves of Madhya Pradesh. International Journal of Veterinary Sciences and Animal Husbandry, 5(4): 152-155. Dhami B, Bhusal A, Neupane B, Nishan KC, Lamichhane S, Bhattarai D and Shrestha B. 2023. Fine-scale habitat characteristics influence the winter habitat use of swamp deer ( Rucervus duvaucelii ) in Shuklaphanta National Park, Nepal. Global Ecology and Conservation , 47(1): 1-11. George SC, Smith TE, Naecana SC, Coleman R and Montgomery WI.2014. Physiological stress in Eurasian badger ( Meles meles ): Effect of host, disease and environment. General and Comparative Endocrinology 200:54-60. Guljari SO, Jorgensen G H M., Eilertsen S, Hansen I, Hagen SB, Floystad I and Palme R. 2019. Measuring faecal glucocorticoid metabolites to assess adrenocortical activity in Reindeer. Animals , 9(11): 987. Hinds JA and Sanchen ER. 2022. The role of the hypothalamus-pituitary-adrenal ( HRA) Axis in test induce anxiety : Assessments physiological responses and molecular details. Stresses 2:146-155. Karaer MC, Cabulj-Kanduce N and Snoj T. 2023. Stress in wildlife: comparison of the stress response among domestic capture and free ranging animals. Frontiers in Veterinary Sciences.DOI 10.3389/fvets.1167016. Konjević, D., Janicki, Z., Slavica, A., Severin, K., Krapinec, K., Želježić, D. And Božić, F. 2016. Monitoring cortisol metabolites in the faeces of captive fallow deer (Damadama L.). Veterinarski Arhiv , 86(3): 363–371. Meetei TR, Sen S. and Meitei A L. 2021. Assessment of the health status of wild ungulate based on body condition evaluation technique in Manipur zoological Garden, Iroisemba, Manipur (India). Journal of Entomology and Zoology Studies , 9(4): 210–213. Mostl, E., Messmann, S., Bagu, E., Robia, C. and Palme, R. (1999). Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. Journal of Veterinary Medicine , 46(10): 621–631. Palme R, Touma C, Arias N, Dominchin MF and Lepschy M. 2013. Steroid extraction: Get the best out of faecal samples. Veterinary Medicine Austria , 100: 238–246. Pavitt AT, Pemberton JM, Kruuk LEB and Walling CA. (2016). Testosterone and cortisol concentrations vary with reproductive status in wild female red deer. Ecology and Evolution, 6(4): 1163–1172. Pérez-Barbería FJ, García AJ, Cappelli J, Landete-Castillejos T, Serrano MP and Gallego L. 2020. Heat stress reduces growth rate of red deer calf: Climate warming implications. PLoS ONE , 15(6):1-26. Potratz EJ, Brown JS, Gallo T, Anchor C and Santymire RM. 2019. Effects of demography and urbanization on stress and body condition in urban white-tailed deer. Urban Ecosystems , 22(5): 807–816. Shah MHA, Rafi U, Yasmeen R and Ahmad M. 2022. Monitoring of cortisol levels in Hog deer with varying environment exposure. International Journal of Innovations in Science and Technology , 4(3): 919–928. Singh KP, Shrivastav, A B, Rajput N, Rokde A, Agrawal S and Gupta N. 2019.Occurrence of Fasciola spp. in free-ranging wild ruminants of Central India. Veterinary Parasitology , 33(2):39-42. Snedecor GW and Cochran WG. 1994. Statistical Methods, 8 th Edn, Iowa state University Press, USA. Soulsby EJL. 1982. Helminths, arthropods and protozoa of domesticated animals, 8th Edn., English Language Society and Bailliere Tindal, London. Whitehouse, KA and Duffus ALJ. 2009. Effect of environmental change of wildlife health. Phil. Trans. R. Soc. Wilcox BA and Colwell RR.2005.Emerging and reemerging infectious diseases: Biocomplexity as an interdisciplinary paradigm. EcoHealth, 2:244-257. 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In India, distributed sub species of barasingha (\u003cem\u003eRucervus duvaucelii branderi\u003c/em\u003e) exclusively found in Kanha tiger reserve of Madhya Pradesh. However, another sub-species of barasingha (\u003cem\u003eRucervus duvaucelii ranjitsinghi\u003c/em\u003e) located in the plains of Brahmaputra River in Assam and North East India and sub species of \u003cem\u003ebarasingha\u003c/em\u003e (\u003cem\u003eRucervus duvaucelii duvaucelii)\u003c/em\u003e found in the fertile plains of Uttar Pradesh including Nepal (Ahmad. 2007; Bajpai \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimplification of ecosystem and urbanization of human colonies beside protected forest areas are major threat to the wildlife conservation enhancing unrest and stressful situations in free ranging wild animals (Wilcox and Colwell, 2005). Depletion in wilderness and encroachment of transitional zones of protected forest area has been building unrest and induce emerging diseases in free ranging wild animals (Whitehouse and Duffus, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Deer family of free ranging habitats has pray based importance in the food chain for big cats helps in shaping the grass land meadows (Singh et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Barasingha serve as important prey for terrestrial mammalian carnivore species while they are agile and sensitive for environmental stressors (Dhami et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStress condition stimulates homeostatic, physiological and behavioral responses linked with joint activity of nervous-endocrine system. Anthropogenic interference in the sylvatic cycle sudden change in the nidus, adverse climatic conditions like drought, rain or wind and territorial fighting injuries act as stimulator for stress hormones (Karaer et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It may induce by many factors and mechanism involving the hypothalamic- pituitary- adrenal (HPA) axis and the sympathetic- adrenal- medullar (SAM) axis. Subsequently the HPA axis is activated leading to release of glucocorticoid hormones while the SAM axis facilitates the release of norepinephrine and epinephrine (Hinds and Sanchen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition to that the short term release of cortisol is helpful in mobilisation of energy to cope with stressor while chronic release of cortisol leads to muscle wastage, tachycardia and reduced immunity and fertility.\u003c/p\u003e\u003cp\u003eIn the present study cortisol accession was evaluated in free ranging barasingha (\u003cem\u003eRucervus duvaucelii branderi\u003c/em\u003e) those were shifted to Satpura and Bandhavgarh tiger reserves with their close vicinity and similar habitat. These herds were also considered for body score evaluation and correlations with faecal cortisol metabolites along with body conditions and intensity of gastrointestinal parasitic loads that directly affect the foraging behaviour.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eThe herd of barasingha in kanha tiger reserve and Satpura tiger reserves were targeted to collect the biological samples using non invasive techniques:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003cp\u003eThe herd of Barasingha were followed early in the morning and observed from a close distance. When the herd moved from their resting place, the freshly laid faecal samples were collected from the pasture. The faecal samples were collected in containers divided into two parts, one part kept without any preservative in cold chain for stress analysis and another part kept in 10% buffered formalin for coprodiagnostic investigation. The samples were brought to school of wildlife forensic and health laboratory, Jabalpur for further processing.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStress evaluation\u003c/strong\u003e\u003cp\u003eThe faecal samples were stored in cold chain and used further dried at 60⁰C for four hours and then powdered finely dispensed in 80% methanol. The mixture vortexed for 30 minutes and centrifuged for 15 minutes at 5000 rpm. The supernatant is collected and stored at -2⁰C according to protocols of Palme et al., (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The supernatant was diluted using assay buffer. The reconstituted diluted samples were run with ELISA assay with following the standard protocol of manufacturer.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCoprodiagnostic Investigation\u003c/strong\u003e\u003cp\u003eThe formalin preserved fresh faecal samples were processed using standard techniques as per Soulsby (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) including qualitative and quantitative techniques and those samples found higher intensity of parasitic load were further used for EPG (egg/oocyst/ gram) counting.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBody condition scoring\u003c/strong\u003e\u003cp\u003eThe animals were photographed with the close vicinity and the body condition was judged based on body evaluation scheme as proposed by Meetei et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The body condition was evaluated based on condition of skin coat, flank ribs, pelvic girdle, vertebral column and lumbar shelf as per the criteria that the animals securing a total score of 0\u0026ndash;5 points were adjudged to be in poor; 6\u0026ndash;8 points in fair and 9\u0026ndash;12 points in good condition.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eSPSS Statistics software version 30.0 was used for analysis of results. Chi square test was done to study the occurrence of gastrointestinal parasites in Barasingha. Independent sample t-test was done to analyse the difference in stress level in Barasingha in both parks. Correlation analysis was performed to evaluate the effect of different parameters on stress level (Snedecor and Cochran, 1982).\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe studies include body evaluation of barasingha of both tiger reserves along with coprodiagnostics and corticosteroids evaluation focussed on foraging behaviour assessment. According to body condition score (BCS) most of the population of barasingha of either sex were found in good body condition (50.0% animals) at Kanha Tiger Reserve (KTR) in comparison to 38.9% Barasingha in Satpura Tiger Reserve (STR). Poor body condition was revealed in 5.5% barasingha of KTR and 8.3% in STR. However, average body condition encountered in 44.4% barasingha of KTR in comparison to 52.8% barasingha of STR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBody condition score evaluation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTiger Reserve\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnimal Examined\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGood\u003c/p\u003e\u003cp\u003e(9\u0026ndash;12 points)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFair\u003c/p\u003e\u003cp\u003e(6\u0026ndash;8 points)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePoor\u003c/p\u003e\u003cp\u003e(0\u0026ndash;5 points)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKanha\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18 (50%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16 (44.44%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e02 (5.55%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSatpura\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14 (38.88%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19 (52.77%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e03 (8.33%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eSkin coat\u0026thinsp;=\u0026thinsp;Rough/smooth, Flank and Ribs\u0026thinsp;=\u0026thinsp;Exposed/Muscular, Pelvic girdle\u0026thinsp;=\u0026thinsp;Exposed/Muscular, Vertebral column and Lumbar\u0026thinsp;=\u0026thinsp;Exposed/ Muscular\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePark wise occurrence of gastrointestinal parasites in Barasingha\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTiger Reserve\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSamples Examined\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIntestinal flukes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStrongyles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCoccidia\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMixed Infection\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKanha tiger reserve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (33%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (11%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2 (6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18 (50%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSatpura tiger reserve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9 (25%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3 (8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e22 (61%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eX\u003csup\u003e2\u003c/sup\u003e, P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.605, 0.605\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.727, 0.674\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.215, 1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.900, 0.477\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSubsequently, qualitative and quantitative coprodiagnostic investigation has also revealed a mean EPG/OPG 1033.33\u0026thinsp;\u0026plusmn;\u0026thinsp;185.592 of Coccidia in KTR and 1205.56\u0026thinsp;\u0026plusmn;\u0026thinsp;192.145 EPG/OPG in Barasingha of STR (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The high rise of stress inducing cortisol was observed in barasingha of KTR and STR those found positive for Coccidiosis (\u003cem\u003eEimeria\u003c/em\u003e spp) followed by Nematodes (Strongyles) and Intestinal flukes (Amphistomes). Similar results were encountered in wild striped hyaena (\u003cem\u003eHyaena hyaena\u003c/em\u003e) population in Southern India with strong correlation of parasitic loads (Ashish, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Bangar, (2019) also emphasized about parasitic load that is responsible for elevation of cortisol metabolites in captive spotted deer (\u003cem\u003eAxis axis\u003c/em\u003e) and Blackbuck (\u003cem\u003eAntelope cervicapra\u003c/em\u003e) in India. However, cortisol level also found increased in captive elephants those have high worm load of flukes and Coccidia (Dhairykar, \u003cem\u003eet al\u003c/em\u003e. 2022).\u003c/p\u003e\u003cp\u003eFurthermore, Mean FCM level was estimated in barasingha as 263.335\u0026thinsp;\u0026plusmn;\u0026thinsp;43.05 pg/ml in KTR and 290.054\u0026thinsp;\u0026plusmn;\u0026thinsp;40.157 pg/ml in STR (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e ). Statistical analysis of cortisol concentration in the feacal metabolites showed non- significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the mean stress level in barasingha of both tiger reserves. In addition to that significant positive correlation between incidence of poor body condition and FCM concentration through Pearson correlation coefficient of 0.575 was calculated in barasingha of KTR and 0.462 in STR. Both values were found highly significant at 99% confidence intervals. It indicates deterioration of body condition coincides with increase in body FCM level. More or less similar findings encountered in Cervidae species (Pavitt et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, significant positive correlation between poor body condition and FCM concentration in barasingha was observed with positive Pearson correlation coefficient of 0.575 in KTR and 0.462 in STR. Both values were found to be highly significant at 99% confidence interval. It indicates deterioration of body condition coincides with increase in body FCM level. Body condition score of an animal is an indicative of its nutritional intake, parasitic load and its impact on health (Sanchez \u003cem\u003eet al.\u003c/em\u003e, 2018). Similar interpretation drawn by George et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) who found elevated faecal cortisol levels in European badger (\u003cem\u003eMelesmeles\u003c/em\u003e) with lower body condition. The authors concluded that poor body condition increases stress in animal and it\u0026rsquo;s susceptibility to be hunted as prey. Hair cortisol may also be used for metabolic estimation (Potratz et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gulzari \u003cem\u003eet al.\u003c/em\u003e 2019) found elevated hair cortisol metabolite concentration in deer family which had lower body condition.\u003c/p\u003e\u003cp\u003eHumidity is a major contributor to thermal stress in animals and it leads to lower body growth (Perez- Barbaria \u003cem\u003eet al.\u003c/em\u003e 2020). However, in the present study more or less similar relative humidity (36\u0026ndash;38%) recorded during the entire study period. Notwithstanding, seasonal variations may consider as a tool for stress evaluation. Higher FCM recorded by Corlatti et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) in Red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e) in Austria during winter compared to summer might be due to low feed intake and lower metabolic rate in summer. Konjevic \u003cem\u003eet al.\u003c/em\u003e (2016) also found higher FCM level in adult Chamois (\u003cem\u003eRupicapra rupicapra)\u003c/em\u003e during summer (186\u0026ndash;3271 ng/g) and winter (430\u0026ndash;2385 ng/g) compared to spring (129\u0026ndash;2896 ng/g). Shah et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) also measured higher urinary and faecal cortisol metabolites in captive hog deer (\u003cem\u003eAxis\u003c/em\u003e porcinus) during summer than winter. However, Mostl et al., (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) emphasized about rain lead to rise in level of FCM that is probably due to increased microbial intake and consequent rise in gut microbial action to metabolize steroids. According to literature available the present study conducted for the first time to evaluate the cortisol and stress building factors in free ranging barasingha of central India. More affords are needed to decrease the stress in such habitat indicator species for their sustenance in the free ranging habitat.\u003c/p\u003e\u003cp\u003eStress in free ranging animals is affected by a myriad of factors including feed intake, environmental conditions. Coccidian parasite affects the health condition of Barasingha pertaining to foraging behaviour and also contributes to higher FCM level. Non- significant difference noted in the mean cortisol level in the two sites. Poor body condition affects stress level as observed in both the tiger reserves.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eNo conflicts of Interest\u003c/strong\u003e: ethical approval and permission from PCCF (Wildlife) Govt. of M.P. was taken for sample collection and procession using non invasive techniques.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Principal Chief Conservator of Forest (Wildlife) for giving permission to collect the biological sample of barasingha from Kanha and Satpura Tiger Reserve, M.P. under project research work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed K. 2007. Ecology and conservation of swamp deer in Terai grasslands of Uttar Pradesh, India. Ph D thesis (Wildlife Science), Aligarh University, India.\u003c/li\u003e\n\u003cli\u003eAshish K, Arora B, Karne D, Kumar V, Khan A S, Umapathy G, Ramesh T and Kalle R. 2023. Glucocorticoid concentration and parasitic load in a wild striped hyaena (\u003cem\u003eHyaena hyaena\u003c/em\u003e) population in Southern India. \u003cem\u003eTheriogenology Wild\u003c/em\u003e, 3(1): 100039.\u003c/li\u003e\n\u003cli\u003eBajpai, S P. 2015. Evaluation of habitat for conserving the hard ground barasingha (\u003cem\u003eCervus duvauceli branderi\u003c/em\u003e Pocock 1943) in Kanha National Park. Ph.D thesis (Botany), Dr. Harisingh Gour Vishwavidyalaya, Sagar.\u003c/li\u003e\n\u003cli\u003eBangar N (2019). Faecal cortisol metabolites as an indicator of stress in captive spotted deer (\u003cem\u003eAxis axis\u003c/em\u003e) and Blackbuck (\u003cem\u003eAntilope cervicapra\u003c/em\u003e) in India. M.V.Sc. \u0026amp; A.H. thesis (Wildlife Health and Management), University of Nairobi. \u003c/li\u003e\n\u003cli\u003eCorlatti L, Palme R, Frey-Roos F and Hackl\u0026auml;nder K. 2011. Climatic cues and glucocorticoids in a free-ranging riparian population of red deer (\u003cem\u003eCervuselaphus\u003c/em\u003e). \u003cem\u003eFolia Zoologica, \u003c/em\u003e60(1): 176 - 180.\u003c/li\u003e\n\u003cli\u003eDhairykar M, Singh KP, Jadav, KK and Rajput N. 2020. Comparison of cortisol level in Asian elephants of different tiger reserves of Madhya Pradesh. International Journal of Veterinary Sciences and Animal Husbandry, 5(4): 152-155.\u003c/li\u003e\n\u003cli\u003eDhami B, Bhusal A, Neupane B, Nishan KC, Lamichhane S, Bhattarai D and Shrestha B. 2023. 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Monitoring of cortisol levels in Hog deer with varying environment exposure. \u003cem\u003eInternational Journal of Innovations in Science and Technology\u003c/em\u003e, 4(3): 919\u0026ndash;928.\u003c/li\u003e\n\u003cli\u003eSingh KP, Shrivastav, A B, Rajput N, Rokde A, Agrawal S and Gupta N. 2019.Occurrence of \u003cem\u003eFasciola \u003c/em\u003espp. in free-ranging wild ruminants of Central India. \u003cem\u003eVeterinary Parasitology\u003c/em\u003e, 33(2):39-42. \u003c/li\u003e\n\u003cli\u003eSnedecor GW and Cochran WG. 1994. Statistical Methods, 8\u003csup\u003eth\u003c/sup\u003e Edn, Iowa state University Press, USA. \u003c/li\u003e\n\u003cli\u003eSoulsby EJL. 1982. Helminths, arthropods and protozoa of domesticated animals, 8th Edn., English Language Society and Bailliere Tindal, London.\u003c/li\u003e\n\u003cli\u003eWhitehouse, KA and Duffus ALJ. 2009. Effect of environmental change of wildlife health. Phil. Trans. R. Soc.\u003c/li\u003e\n\u003cli\u003eWilcox BA and Colwell RR.2005.Emerging and reemerging infectious diseases: Biocomplexity as an interdisciplinary paradigm. EcoHealth, 2:244-257.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Stress evaluation, Faecal Cortisol, Barasingha, Body condition score, Kanha Satpura, ELISA","lastPublishedDoi":"10.21203/rs.3.rs-7252608/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7252608/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBarasingha kwon as hard ground swamp deer (\u003cem\u003eRucervus duvaucelii branderi\u003c/em\u003e) found exclusively in Kanha Tiger Reserve of Madhya Pradesh while small herd where shifted to Satpura and Bandhavgarh tiger reserves under ex-situ conservation plan. Barasingha enlisted in threatened species of Schedule-I and only three sub-species existing in India. During health monitoring and disease diagnosis schedule, physical body score evaluation and disease diagnostics of free ranging barasingha were performed using non invasive techniques. Freshly voided fecal pallets of barasingha were used for two way analysis (i) coprodiagnostic analysis to screen the gastrointestinal parasitic occurrence (ii) assessment of faecal cortisol metabolites using ELISA diagnostic kit (DetecX, Arbor assay, Ann Arbor, Michigan, USA). The extracted cortisol aliquots were analyzed and obtained mean fecal cortisol metabolites (FCM) level 263.335\u0026thinsp;\u0026plusmn;\u0026thinsp;43.050 pg/ml and 290.054\u0026thinsp;\u0026plusmn;\u0026thinsp;40.147pg/ml in Barasingha of Kanha and Satpura tiger reserve respectively. Significant positive correlation was observed between FCM concentration with positive fecal samples of \u003cem\u003eEimeria\u003c/em\u003e spp in barasingha of both the parks at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 level (2- tailed). Furthermore, significant positive correlation was also observed between FCM level and poor body condition score in Barasingha at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 level (2- tailed). The study instigate poor body condition has strong correlation with coccidiosis that increases corticosteroids in barasingha affecting foraging behaviour.\u003c/p\u003e","manuscriptTitle":"Coccidiosis induces stress hormones in hard ground Barasingha (Rucervus duvaucelii branderi)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 23:40:02","doi":"10.21203/rs.3.rs-7252608/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80530c10-0fa2-4700-ae4f-228ababa3860","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-15T13:49:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 23:40:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7252608","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7252608","identity":"rs-7252608","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}