Experimental Foot-and-mouth disease virus (FMDV) infection induces reversible exocrine pancreatic insufficiency and irreversible endocrine pancreatic insufficiency in Holstein-Friesian crossbred heifers

Experimental Foot-and-mouth disease virus (FMDV) infection induces reversible exocrine pancreatic insufficiency and irreversible endocrine pancreatic insufficiency in Holstein-Friesian crossbred heifers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Experimental Foot-and-mouth disease virus (FMDV) infection induces reversible exocrine pancreatic insufficiency and irreversible endocrine pancreatic insufficiency in Holstein-Friesian crossbred heifers Padmanaban Udhayabanu, Priyanka Mahadappa, Karikalan Mathesh, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7157311/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 As hyperglycemia is a consistent finding in cattle recovered from FMD, we hypothesized that damage to the pancreas by FMD virus (FMDV) is the basis for the alterations in energy metabolism. Accordingly, crossbred heifers were either inoculated with virulent FMDV serotype O by intradermolingual (n = 7) or mock-infected with saline (n = 7). Blood samples were collected on day 0, 7, 14, 30, 60, 90, 120, 150, and 180 post-infection (dpi) to determine the concentrations of exocrine pancreatic enzymes, insulin, and indicators of energy metabolism. The results indicated a significant elevation in the serum amylase, lipase, and TLI by dpi 7 in FMDV-infected heifers that returned to baseline by dpi 60–120. In contrast, decrease in the serum insulin, first recorded on dpi 14, persisted till dpi 180 (P < 0.0001). The circulating concentrations of glucose, glycated hemoglobin, triglycerides, NEFA and BHB showed a significant increase by dpi 30 and did not recede to basal level till dpi 180 in the FMDV-infected group (P < 0.0001). Pancreatic histopathology and immunohistochemistry in a heifer that died on 12 dpi revealed necrotic and inflammatory changes in the pancreas with intense immunolabeling for FMDV antigens both in the pancreatic acini and islets. In another heifer that died on dpi 49, regenerative changes were found in the exocrine pancreas, while necrotic changes persisted in the islets of Langerhans, with moderate FMDV antigen labeling. It was concluded that experimental FMDV infection-induced endocrine pancreatic insufficiency was irreversible, whereas exocrine pancreatic function could be restored by dpi 120. Amylase Foot-and-mouth Disease Insulin Lipase Trypsin-like immunoreactivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Foot-and-mouth disease (FMD) is one of the most highly contagious viral diseases affecting cloven-hoofed animals, including cattle, buffalo, swine, sheep, goats, bison, and deer worldwide. Due to its broad host range, rapid transmission, and significant economic impact, the OIE (2000) has classified FMD as the most dangerous disease affecting animals. The economic consequences of FMD vary by region; in enzootic areas, it causes major losses due to reduced productivity and trade restrictions, with estimated annual costs ranging from $ 6.5 to $ 21 billion (Alhaji et al., 2020 ). In regions or countries free of FMD, outbreaks can lead to losses exceeding $ 1.5 billion annually (Knight-Jones and Rushton, 2013 ). Although most affected adult animals recover clinically within 2–3 weeks of infection, they often do not regain their productivity and reproductive efficiency for a long period. In fact, 10–20% of animals affected by FMD may permanently lose some of their productivity (Kitching, 2002 ). Key concerns for animals recovering from FMD in enzootic areas include reduced milk production (Bayissa et al., 2011 ), poor fertility (Chaters et al., 2018 ), decreased draft capacity (Perry and Randolph, 2003 ), and inadequate weight gain (Rufael et al., 2008 ). The pancreas, an endocrine and exocrine gland, plays a vital role in digesting and metabolizing carbohydrates, fats, and proteins. In bovines, productive and reproductive functions depend on effective metabolism (Laskowski et al., 2016 ). Metabolic challenges can lead to lower productivity, reproductive issues, and decreased fertility in ruminants (De Koster and Opsomer, 2013 ; Laskowski et al., 2016 ). The pancreas is known to serve as a preferred site for the replication of the foot-and-mouth disease virus (FMDV) in adult mice (Sanz-Ramos et al., 2008 ) and as a site for persistence in infected cattle (Alexandersen et al., 2002 ). FMDV in pancreatic tissue may damage the islets of Langerhans and pancreatic acinar cells, ultimately causing pancreatic dysfunction. Several studies have observed injury to pancreatic acinar cells and islets of Langerhans during the acute phase of FMD in cattle (Sutmoller et al., 2003; Ghanem and Abdel-Hamed, 2010; Sahoo et al., 2023 ). However, research on the long-term effects of FMDV infection on the function of both the exocrine and endocrine pancreas is currently limited. Understanding these short-term and long-term effects is essential for developing therapies or remedial measures to mitigate production and reproduction losses in cattle. Therefore, this study was designed to evaluate the short-term and long-term effects of experimental FMDV infection on the endocrine and exocrine pancreatic functions in Holstein-Friesian crossbred heifers. Materials and Methods Animals Fourteen apparently healthy Holstein Friesian (HF) crossbred heifers, aged 8 to 10 months, were selected for this study. None of these heifers had a history of previous FMDV infection, nor had been vaccinated against the virus. The heifers were dewormed and quarantined for a period of four weeks for acclimatization. They were screened for the presence of FMDV antibodies using an in-house ELISA kit developed by ICAR-IVRI, Bengaluru, which detects FMDV non-structural proteins (NSP). Animals that did not show the presence of NSP antibodies were further examined for virus neutralization test (VNT). Only those animals that exhibited a virus neutralization (VN) titer of ≤8 (considered seronegative) were included in the study. The heifers were then randomly inoculated with FMDV serotype O by intradermolingual (n=7) or mock-infected with saline (n=7). Ethical approval The experimental protocol received ethical approval from the Institutional Animal Ethics Committee of the ICAR - Indian Veterinary Research Institute in Bengaluru, as well as from the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under reference number V-11011(13)/13/2023-CPCSEA-DADF, dated January 5, 2024. The experiment was conducted in compliance with CPCSEA regulations. Experimental infection of animals A bovine-derived FMDV serotype O/IND/R2/75 was used for experimental infection. Heifers were inoculated with 4.0 log 10 BID 50 (50% bovine tongue infectious dose) of virus suspended in 0.2 mL of media via intradermolingual route. 0.2 mL of sterile normal saline was injected by intradermolingual route into mock-infected animals. Both FMDV-infected and mock-infected animals were housed separately in the biocontainment facility under iso-managerial conditions. Clinical observations were recorded from 2 days before the experimental infection to 180 days post-infection (dpi). Confirmation of FMDV infection and serotype Confirmation of foot-and-mouth disease virus (FMDV) infection in the animals was performed using an in-house serotype-differentiating antigen detection ELISA kit developed by the Directorate of Foot-and-Mouth Disease in India, as outlined by Bhattacharya et al. (1996). The serotype-differentiating antigen detection ELISA performed using samples from the epithelium of the tongue or the interdigital space confirmed that the infection was caused by serotype O of the FMD virus (FMDV). Sample collection Blood samples were collected on the day of infection (day 0) and at various time points following the experimental infection, namely, 7, 14, 30, 60, 90, 120, 150, and 180 dpi. Approximately 10 mL of blood was drawn from the jugular vein of each animal using a vacutainer coated with heparin (BD, Franklin, USA) as an anticoagulant for plasma samples. Additionally, another 10 mL of blood was drawn into vacutainers containing a clot activator (BD, Franklin, USA) for serum collection. Each vial was labelled with the animal number, date, and time of collection. The tubes were centrifuged at 3000g for 20 min to clarify the samples. Following centrifugation, the serum and plasma were transferred to fresh 4 mL Eppendorf tubes and stored at -20°C for further analysis. Biochemical analysis Plasma glucose, serum amylase, lipase, triglycerides, and cholesterol were estimated by colorimetric method using commercial kits (GenX, Proton Biologicals, India) and a Photometer version NP80 (Sl No. M81334; Implen GmbH, Germany). Serum insulin was estimated using bovine enzyme immunoassay (EIA; Mercodia, Winston Salem, NC; with the sensitivity of ≤ 0.025 µg/L). Plasma NEFA concentration was estimated by colorimetric method (Clementia Biotech, New Delhi, India, Sensitivity: 0.05 mmol/L, Detection range: 0.05-2.0 mmol/L; Inter assay CV: 1.55%, Intraassay CV: 0.6%). Plasma BHB concentration was estimated by colorimetric method (Abcam, ab83390, Sensitivity: 0.01 mM, Range: 0.01 mM - 0.2 mM, Inter assay CV: 1.55%, Intraassay CV: 0.6%). Trypsin-like immunoreactivity (TLI) was estimated by Canine specific TLI (Trypsin-like immunoreactive protein) ELISA kit (Catalog # ELK 0307, ELK Biotechnology Co. Ltd., India). The analytical sensitivity of TLI kit was 11.3 ng/ml with a detection limit of 31.25-2000 ng/ml. The manufacturer reported intra-assay and inter-assay CV (=SD/mean×100) were <8% and <10% respectively. Glycated haemoglobin was estimated by ion exchange chromatography method (Coral clinical systems, India). Histopathology and immuno-histochemistry Out of seven experimentally infected HF crossbred heifers, one heifer died on dpi 12, and another died on dpi 49. A necropsy examination was conducted, and pancreatic tissues were collected in 10% neutral buffered formalin for histopathology and immunohistochemistry following standard guidelines (Luna, 1968; Ramos-Vara, 2017). Statistical analysis A two-way repeated measures ANOVA was conducted to evaluate the time-dependent effects of FMDV infection on the endocrine pancreas, exocrine pancreas, and energy metabolism at various days post-infection. A Bonferroni test was applied to compare the pairwise mean differences between different days of FMDV infection. A significance level was set at 5%. Data analysis and chart preparation were performed using GraphPad Prism version 9.5.4. Results Onset of hyperpnea and pyrexia was recorded by 48 h post-inoculation of FMDV; excessive salivation, vesicular lesions in the oral cavity, and nasal discharge appeared by 72 h post-inoculation. Vesicular lesions at the interdigital space were recorded by 96 h post-inoculation (Supplementary figure 1). Oral lesions were the first to heal, while hoof lesions healed by two weeks post-inoculation. After the apparent clinical convalescence by 3 - 4 weeks from the acute lesion, FMDV infected heifers developed ruffled hair coat with hypopigmented hairs in the trunk area around 5-6 week (3/7) and cracked hooves around 6–7-week post-inoculation (4/7). Onset of panting was consistently observed around 8 - 9 weeks post-inoculation in all the FMDV-infected heifers (7/7) (Supplementary Figure 2). During the acute phase, a significant increase in the concentrations of serum amylase, lipase, and TLI was observed on dpi 7 (Figure 1A, 1C, and 1E) in FMDV-infected heifers compared to mock-infected heifers. Elevated concentrations of amylase and TLI returned to pre-infection concentrations by dpi 60 (Figure 1D and 1F), while that of lipase was on dpi 120. Changes in the serum amylase and lipase indicated their potential utility as a biomarker during the first 60 days of FMD infection. In striking contrast to exocrine pancreatic enzymes, serum concentrations of insulin showed a significant decrease by dpi 14 (Figure 1G) in FMDV-infected heifers that persisted till dpi 180 (Figure 1H). Among the indicators of energy metabolism, serum triglycerides, plasma non-esterified fatty acids (NEFA), and plasma β-hydroxybutyrate showed a significant increase by dpi 14 (P<0.01; Figure 2E, 2G, and 2I) in the FMDV infected heifers and remained elevated till dpi 180 (P0.05; Figure 2A and 2C) between the FMDV-infected heifers and mock-infected group. Onset of hyperglycemia in the FMD-infected heifers was apparent by dpi 30 while, glycated Hb (%) showed a significant rise by dpi 90 (Fig. 2D). Similar to lipids, plasma glucose and glycated Hb (%) remained elevated till dpi 180 in the heifers (P<0.0001; 2B and 2D). The results are further supported by the histopathological and immunohistochemical findings in the pancreas of heifers that died on dpi 12 (Heifer #A681) and dpi 49 (Heifer #A687). Histopathology of the pancreas revealed necrosis and mononuclear infiltration of the acinar cells (Fig. 3 A and B) and Langerhans (Fig. 3 D and E) in the heifer that succumbed on dpi 12. Intense immunolocalization of the non-structural protein 3A of FMDV suggested colonization of FMD and possible viral replication in the exocrine (Fig. 3C) and endocrine (Fig. 3F) pancreas. In contrast to the regenerative changes in the acinar cells (Fig. 4A), degenerative and necrotic changes persisted in the Islet cells of the pancreas (Fig. 4C) in the heifer that died on dpi 49. Though moderate immunostaining of the non-structural protein 3A of FMDV indicated persistence of FMDV in the exocrine and endocrine pancreas till dpi 49 (Fig. 4 B and D). Discussion The clinical signs observed during both the acute and chronic phases of FMDV infection in the HF crossbred heifers align with previous findings (Ghanem and Ahmed, 2010; Kamal et al., 2018). Symptoms such as fever, excessive salivation, rapid breathing, vesicular and ulcerative lesions in the oral cavity and interdigital space can be linked to the viraemic phase, during which FMDV replicates in the oropharyngeal epithelium and spreads systemically to other tissues. This replication causes vesicular and ulcerated lesions in both the mouth and interdigital space of the feet (Sutmoller et al., 2003). Chronic signs like panting, rough hair coat, lameness, and overgrown hooves are consistent with previous reports in Egyptian cattle, where these symptoms recorded three months after an FMD outbreak (Ghanem and Ahmed, 2010). Panting and rough hair coat observed could be imputed to the alterations in the metabolic functions (Ghanem and Ahmed, 2010). Chronic lameness might lead to improper wear of the hooves, resulting in overgrown hooves (Belsham et al., 2024). Chronic signs such as cracked hooves with partial loss of black pigmentation in the hair coat have not been reported elsewhere. Exocrine pancreatic function We recorded the trend of circulating concentrations of serum amylase, lipase, and TLI from dpi 0 to 180 day post-FMDV infection to assess the temporal changes in exocrine pancreatic damage, which is often challenging to diagnose in ruminants (Vargas-Rodriguez et al., 2014; Guo et al., 2021). Since amylase is also produced by rumen microflora (Vargas-Rodriguez et al., 2014), and salivary glands contribute to lipase, a significant increase in two or more digestive enzymes such as amylase, lipase, and TLI indicates pancreatic injury or inflammation in ruminants. In cows infected with FMDV, serum levels of amylase and lipase increased significantly during dpi 7-30 and dpi 7-90, respectively (Fig. 1 A to D). Previous studies have also reported elevated levels of amylase and lipase in cattle infected with FMDV (Kamal et al., 2018; Soltani et al., 2020). Additionally, a notable rise in serum TLI levels was observed between dpi 7-14 in FMDV-infected cows, suggesting damage to the pancreatic acini (Flamion et al., 1987). Trypsin is released into circulation when these acinar cells are damaged, leading to increased TLI levels. This study is the first to report elevated TLI in FMDV-infected cattle, though it is a diagnostic marker for acute pancreatitis in humans, dogs, and cats (Flamion et al., 1987; Xenoulis, 2015). Elevated amylase, lipase, and TLI in FMDV-infected HF crossbred heifers may be due to damage caused by FMDV infection in exocrine pancreatic acinar cells. This is supported by previous studies that have extensively documented degeneration and necrotic changes in the pancreatic acinar cells during FMDV infection in cattle (Yeotikar et al., 2003; Ghanem and Abdel-Hamid, 2010; Nahed et al., 2010; Artz et al., 2011; Sahoo et al., 2023). Similar pathological findings were observed in the exocrine pancreas of an HF crossbred heifer that succumbed to experimental FMDV infection on dpi 12 in the present study (Figure 3A). Intense immunostaining of the 3A protein of FMDV on dpi 12 (Fig. 3C) in the pancreatic acinar cells (Heifer #A681) indicated a high viral load. It is reported that FMDV has a special affinity for pancreatic tissue and can replicate there, leading to inflammatory changes in the pancreas (Alexandersen et al., 2002; Sutmoller et al., 2003; Sanz-Ramos et al., 2008). The serum concentrations of exocrine pancreatic enzymes returned to pre-infection levels by dpi 60 to 90, indicating resolution of inflammation. This is supported by the regenerative changes observed in the pancreatic acinar cells of the HF crossbred heifer that succumbed on dpi 49 (Fig. 4A). Endocrine pancreatic function Experimental infection of the heifers with FMDV resulted in ~20% reduction in the serum insulin concentrations by dpi 14 that further decreased to ~40% by dpi 30 dpi compared to mock-infected heifers (Fig. 1 G and H). Seminal experiment by Barbni et al. (1966) demonstrated induction of diabetes mellitus in the FMD infected cattle. Hypoinsulinemia was reported in cattle after recovery from natural FMD infection (Nahed, 2010). Our unpublished observations indicate insulin insufficiency in the purebred HF and Jersey cows till day 180 post-FMD outbreak. A significant reduction in serum insulin concentrations in FMDV-infected HF crossbred heifers might be due to virus-induced destruction of insulin-secreting β-cells in the pancreas as evidenced by the presence of FMDV antigens in the endocrine pancreas and absence of regenerative changes in the endocrine pancreas of a heifer that died on day 49 post-infection (Fig. 4 C and D). Similar to the bovine viral diarrhea virus, FMDV has also been associated with the development of type 1 diabetes in cattle (Nahed, 2010). FMDV can directly damage insulin-producing pancreatic β-cells through viral replication (Barbni et al., 1966; Sanz-Ramos et al., 2008) or through autoimmune reactions triggered by the virus. However, none of the experimentally infected HF crossbred heifers in this study developed glycosuria, indicating insulin insufficiency rather than insulin deficiency. In contrast to the resolution of exocrine pancreatic damage around dpi 120 as evidenced by normal concentrations of pancreatic enzymes (Fig. 1 B, D, and F), decreased insulin concentration persisted till dpi 180 (Fig. 1H) in the FMDV-infected HF crossbred heifers. Presence of FMDV antigens and absence of regenerative changes in the endocrine pancreas by dpi 49 in a heifer #A687) also support the insulin insufficiency till dpi 180. Collectively, the exocrine pancreas function was restored by dpi 90, while endocrine dysfunction persisted until dpi 180. Energy metabolism A significant increase in the plasma glucose by dpi 30 in FMDV-infected HF crossbred heifers (Fig. 2B; P < 0.0001) might be due to insulin insufficiency. Hyperglycemia is a consistent finding in cattle (Yeotikar et al., 2003; Gokce et al., 2004; Bakrakati et al., 2015; Kamal et al., 2018; Soltani et al., 2020) and sheep (Gattani et al., 2011) recovered from FMD. Glycated hemoglobin, a fraction of hemoglobin that binds to plasma glucose, serves as a marker for evaluating glucose levels over an extended period (Neumann, 2020; Norris and Schermerhorn, 2022). A significant increase (P < 0.0001) in glycated hemoglobin levels observed in FMDV-infected HF crossbred heifers from 90 to 180 dpi, can be attributed to the chronic hyperglycemia present in these animals. Additionally, hypertriglyceridemia from dpi 14 to 180 in the FMDV-infected heifers than in mock-infected heifers (Fig. 2 E and F; P < 0.0001), indicates impaired insulin action. Insulin typically activates lipoprotein lipase to liberate fatty acids from the triglycerides. Therefore, FMDV-induced insulin insufficiency might have disrupted the activation of lipoprotein lipase, resulting in the accumulation of triglycerides. Our findings are in agreement with Soltani et al. (2020), who also reported hypertriglyceridemia in animals affected by FMD. A significant increase in the plasma concentrations of NEFA and BHB from dpi 14 to 180 (Fig. 2 I and J; P=0.0004) may be linked to hypoinsulinemia in the FMDV-infected heifers. During hypoinsulinemia, the body mobilizes subcutaneous fat, leading to the production of NEFA (Adewuyi et al., 2005). Hypoinsulinemia also promotes the entry of NEFA into mitochondria, which in turn facilitates the formation of ketone bodies such as BHB. Our findings are consistent with earlier reports (Kamal et al., 2018) that indicated elevated levels of NEFA and BHB in animals affected by FMDV. Histopathology and immunohistochemistry of the pancreas The major histopathological findings observed in the HF crossbred heifer that died on the 12 th day post-FMDV included diffuse necrosis of the acinar cells, along with the infiltration of mononuclear cells such as lymphocytes, macrophages, and plasma cells in the exocrine pancreas. Additionally, degeneration and necrosis of the islets of Langerhans in the endocrine pancreas were noted. Similar pancreatic pathology has been documented in previous studies involving FMD-affected calves (Sahoo et al., 2023), cattle (Barker et al., 1992), and gazelles (Berkowitz et al., 2010). These findings are supported by elevated concentrations of circulating pancreatic enzymes, including amylase, lipase, and TLI, observed in FMD-infected HF crossbred heifers from dpi 7 to 120 (Fig. 1 A to F). Elevated pancreatic enzymes are also reported during the acute phase of FMD (Kamal et al., 2018; Soltani et al., 2020). Intense cytoplasmic immunolabeling for FMDV antigen in the islets of Langerhans indicated colonization and possible replication of the virus in these areas. The pancreas is known to be the preferred site for viral replication in adult mice, with the highest viral load detected 24 h post-FMDV infection (Sanz-Ramos et al., 2008). Similar microscopic lesions, such as lymphocytic infiltration in both pancreatic acinar cells and islets of Langerhans, necrosis of acinar cells and islets of Langerhans were noticed in the mice infected with FMDV (Sanz-Ramos et al., 2008). Several reports suggest that FMDV can replicate in the pancreatic tissue of cattle, resulting in necrosis and degeneration of both the endocrine and exocrine pancreas (Alexandersen et al., 2002; Kamal et al., 2018). In contrast, histopathological examination of the pancreatic tissue from another FMDV-infected HF crossbred heifer that died on dpi 49 revealed regenerative changes in the exocrine pancreas. These observations align with the normal levels of pancreatic enzymes, such as amylase, lipase, and TLI, found in FMDV-infected HF crossbred heifers by 120 dpi in the current study. However, necrotic changes in the islets of Langerhans persisted in the endocrine pancreas, which corresponds with persistently decreased concentrations of serum insulin in experimentally infected HF crossbred heifers. Mild to moderate cytoplasmic immunolabeling for FMDV antigen was noted in both acinar cells and the islets of Langerhans, indicating the persistence of FMDV in the pancreas. This notion is supported by Alexandersen et al. (2002), who suggested that FMDV can persist in the pancreas along with other organs such as the mammary gland, testicles, pituitary gland, and thyroid for extended periods. Furthermore, Arzt et al. (2011) indicated that disruption of pancreatic function accounts for the hyperglycemia observed in cattle that have recovered from FMD. Conclusion Experimental FMDV infection induced reversible damage to exocrine pancreas while irreversible damage to the endocrine pancreas. Declarations Acknowledgements The authors would like to thank the Director and Joint Director of ICAR – Indian Veterinary Research Institute, Regional Campus, Bengaluru for the necessary support. The authors thank the Department of Biotechnology, Ministry of Science and Technology and Government of India for the financial support received for this work. Funding The project was funded by Department of Biotechnology, Ministry of Science and Technology, Government of India to Dr. Priyanka Mahadappa vide grant No.BT/PR30711/BIC/101/1135/2018. Conflict of Interest Statement The authors have no relevant financial or non-financial interests to disclose Author Contributions Priyanka Mahadappa conceptualized the study.All authors contributed to the study design. Material preparation, data collection and analysis were performed by Padmanaban Udhayabanu, Priyanka Mahadappa, Karikalan Mathesh, Saravanan Paramasivam, Aadhithya Muthuswamy, Umapathi V, B H Manjunatha Patel, Veerakyathappa Bhanuprakash, Dechamma Hosuru Joyappa, Pallab Chaudhuri, and Narayanan Krishnaswamy. The first draft of the manuscript was written by Priyanka Mahadappa, Narayanan Krishnaswamy, and Padmanaban Udhayabanu. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Adewuyi AA, Gruys E, van Eerdenburg FJCM (2005) Non esterified fatty acids (NEFA) in dairy cattle. A review. Vet Q 27:117–126 Alexandersen S, Zhang Z, Donaldson AI (2002) Aspects of the persistence of foot-and-mouth disease virus in animals—the carrier problem. 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Sutmoller P, Barteling SS, Olascoaga Raul Casas and Sumption KJ (2003) Control and eradication of foot-and-mouth disease. Virus Res 91:101–144 Sutmoller P, Casas OR (2002) Unapparent foot and mouth disease infection (sub-clinical infections and carriers): implications for control. Rev Sci Tech 21:519–529 Vargas-Rodriguez CF, Engstrom M, Azem E, Bradford BJ (2014) Effects of dietary amylase and sucrose on productivity of cows fed low-starch diets. J Dairy Sci 97:4464–4470. WOAH (2024) WOAH Terrestrial Manual 2022. In: The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual), 13th edn. Xenoulis PG (2015) Diagnosis of pancreatitis in dogs and cats. Journal of Small Animal Practice 56:13–26. https://doi.org/https://doi.org/10.1111/jsap.12274 Yeotikar P V, Bapat ST, Bilolikar SC, Kulkarni SS (2003) Metabolic profile of healthy cattle and caftle affected by foot‐and‐mouth disease. Vet Rec 153:19–20 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7157311","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489309181,"identity":"e6b3712a-f592-46c1-8084-411017c8ca03","order_by":0,"name":"Padmanaban Udhayabanu","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Padmanaban","middleName":"","lastName":"Udhayabanu","suffix":""},{"id":489309182,"identity":"bb857077-c830-4894-8221-3fe95f5bc251","order_by":1,"name":"Priyanka Mahadappa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACA2YGAzCDH0QkFJCgRUKyAaTFgBgtDFAtBgegXILAnJ154+fKNps64/OrEz88MGCQ5xc7gF+LZTNbseTZtjQJsxtvN0sAHWY4c3YCAYcd5jGQbDhzGKjl7AaQlgSD24S1GP9sOPNfwnjG2c0/iNViJtlQcUDCgL93G3G2AP1SZtlQkSw54wbvNosEAwnCfjHnP7z5ZoOBHT9//9nNN39U2MjzSxPQggASYJUSxCoHAf4DpKgeBaNgFIyCkQQAXPxBD+4AVpwAAAAASUVORK5CYII=","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Mahadappa","suffix":""},{"id":489309183,"identity":"ecaf1d74-65c8-45b4-980a-522f8ac52bf6","order_by":2,"name":"Karikalan Mathesh","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Karikalan","middleName":"","lastName":"Mathesh","suffix":""},{"id":489309184,"identity":"69a972a7-8442-4075-877e-40d076c0340f","order_by":3,"name":"Saravanan Paramasivam","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Saravanan","middleName":"","lastName":"Paramasivam","suffix":""},{"id":489309185,"identity":"9816b79c-852e-4ba0-8350-d6675b19fd56","order_by":4,"name":"Aadhithya Muthuswamy","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Aadhithya","middleName":"","lastName":"Muthuswamy","suffix":""},{"id":489309186,"identity":"eb32ca03-20ac-44c7-9c57-7fd290250f93","order_by":5,"name":"V Umapathi","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"V","middleName":"","lastName":"Umapathi","suffix":""},{"id":489309187,"identity":"7e974030-3d7f-4c3c-b838-16160260a818","order_by":6,"name":"B H Manjunatha Patel","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"B","middleName":"H Manjunatha","lastName":"Patel","suffix":""},{"id":489309189,"identity":"575b9539-9124-4795-b15d-bfc1275f3e52","order_by":7,"name":"Veerakyathappa Bhanuprakash","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Veerakyathappa","middleName":"","lastName":"Bhanuprakash","suffix":""},{"id":489309191,"identity":"0fa7bb0a-3a99-4117-8adb-aeeb1b0e7026","order_by":8,"name":"Dechamma Hosuru Joyappa","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Dechamma","middleName":"Hosuru","lastName":"Joyappa","suffix":""},{"id":489309192,"identity":"8ae983f5-4f41-4203-a74c-c87560ddf600","order_by":9,"name":"Aniket Sanyal","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Aniket","middleName":"","lastName":"Sanyal","suffix":""},{"id":489309193,"identity":"003ca0b1-1c0c-4054-ae62-13fdb60feb5e","order_by":10,"name":"Pallab Chaudhuri","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Pallab","middleName":"","lastName":"Chaudhuri","suffix":""},{"id":489309195,"identity":"ccba3e57-f3d6-4e59-b4fc-5e6c501d7a7a","order_by":11,"name":"Narayanan Krishnaswamy","email":"","orcid":"","institution":"ICAR - Indian Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Narayanan","middleName":"","lastName":"Krishnaswamy","suffix":""}],"badges":[],"createdAt":"2025-07-18 11:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7157311/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7157311/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87401835,"identity":"a6fe0cba-789a-4c8a-b970-a505271e06dd","added_by":"auto","created_at":"2025-07-23 11:58:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental FMDV infection induces acute and chronic exocrine and endocrine pancreatic insufficiency in the HF crossbred heifers. \u003c/strong\u003eHolstein Friesian (HF) crossbred heifers of 8 to 10 months were inoculated with 1.0×10\u003csup\u003e4\u003c/sup\u003e 50% bovine tongue infectious dose (BTID\u003csub\u003e50\u003c/sub\u003e) of FMD virus suspended in 0.2 mL media by intra-dermo lingual route (Day 0 post-infection, dpi 0). Sera were harvested on dpi 0, 7, and 14 to determine the acute effects of FMDV infection on the pancreatic infection. To determine the chronic effects, sera were collected at a monthly interval from dpi 30 to 180. Serum concentrations of amylase and lipase were estimated by a colorimetric method while that of TLI and insulin were assayed by ELISA kits. Data were analyzed by mixed model ANOVA by fitting infection status, dpi, and their interaction as fixed effects and heifer as random effect. Heifer variation contributed from 2 to 31% of unexplained variation for all the pancreatic function indicators. Line charts in the left column indicate the acute changes (Fig. 1A, 1C, 1E, and 1G), while the right column charts represent the chronic changes (Fig. 1B, 1D, 1F, and 1H). Onset of experimental FMDV infection induced exocrine pancreatic damage was recorded by dpi 7 as revealed by a significant increase in the serum amylase, lipase, and TLI (Fig. 1A, 1C, 1E); however, damage to Islets of Langerhans was apparent by dpi 14 (Fig. 1G). Further, all the pancreatic function indicators were remained elevated significantly till dpi 30. By dpi 60, the concentration of amylase, and TLI was comparable to that of dpi 0 and the values of mock-infected group on dpi 60 (P\u0026gt;0.05; Fig. 1B and 1F), while that of lipase returned to physiological range by dpi 120 (Fig. 1D). In contrast, insulin insufficiency recorded on dpi 14 persisted till dpi 180 suggesting irreversible injury to the islets of Langerhans by FMDV infection.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/4cc77953a1ad2f1fc743ce14.png"},{"id":87401837,"identity":"9a15ae5b-aefb-4315-95ac-284db8f8af7b","added_by":"auto","created_at":"2025-07-23 11:58:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcute and chronic effects of experimental FMDV infection on the indices of energy metabolism in the HF crossbred heifers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHolstein Friesian (HF) crossbred heifers of 8 to 10 months were inoculated with 1.0×10\u003csup\u003e4\u003c/sup\u003e 50% bovine tongue infectious dose (BTID\u003csub\u003e50\u003c/sub\u003e) of FMD virus suspended in 0.2 mL media by intra-dermo lingual route (Day 0 post-infection, dpi 0). Sera were harvested on dpi 0, 7, and 14 to determine the acute effects of FMDV infection on the indicators of energy metabolism. To determine the chronic effects, sera were collected at monthly interval from dpi 30 to 180. Plasma concentrations of glucose, NEFA, and BHB and serum triglycerides were estimated by a colorimetric method. The concentration of HbA1c in the hemolyzed blood was determined by ion exchange chromatography method. Data were analyzed by mixed model ANOVA by fitting infection status, dpi, and their interaction as fixed effects and heifer as random effect. When the interaction effect was non-significant, main effects model was considered. Heifer variation was \u0026lt;15% for all the indicators of energy metabolism during acute phase; however, during chronic phase of FMD infection, it was \u0026lt;23% for all the parameters except BHB for which it was 40%. Line charts in the left column indicates the acute changes (Fig. 2A, 2C, 2E, 2G, and 2I), while the right column charts represent the chronic changes ((Fig. 2B, 2D, 2F, 2H and 2J). A significant increase in the serum triglycerides, plasma NEFA and BHB by dpi 14 (P\u0026lt;0.05; Fig.\u0026nbsp; 2E, 2G, and 2I) suggested the onset of dyslipidemia in the FMD-infected group; however, the plasma glucose and Hb1Ac remained comparable (P\u0026gt;0.05; Fig. 2A, and 2C). Onset of hyperglycemia and hyperglycation of Hb was apparent by dpi 30 and 90 and remained elevated till dpi 180 (P\u0026lt;0.05; (P\u0026gt;0.05; Fig. 2A, and 2C). Further, elevation of serum triglycerides, NEFA and BHB, which was first observed on dpi 14, persisted till dpi 180 in the FMD-infected heifers (Fig. 2F, 2H, and 2J). Collectively, the results indicated that FMDV infection induced hyperglycemia and dyslipidemia in the HF crossbred heifers.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/673c7019f334590586929886.png"},{"id":87401840,"identity":"b11bbaf7-1b7b-4729-9d52-5529bb87be2f","added_by":"auto","created_at":"2025-07-23 11:58:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5589420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental FMDV induced changes in the pancreas of a HF crossbred heifer (#A681) that succumbed on day 12 post-infection.\u003c/strong\u003e Representative photomicrographs of exocrine (a, b, and c) and endocrine pancreas (d, e, and f) showing degenerative and infiltrative changes.\u003c/p\u003e\n\u003cp\u003eA. Diffuse acinar cell necrosis with loss of acinar architecture and infiltration of mononuclear cells (arrow mark). H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eB. Higher magnification of Fig 3A. showing severe infiltration of lymphocytes in the interlobular area (asterisk). H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eC. Note intense cytoplasmic immunolabeling of non-structural protein of FMDV 3A in the acinar cells (arrow mark) and Langerhans cells (asterisk). IHC\u003c/p\u003e\n\u003cp\u003eD. Severe degeneration and necrosis of islets of Langerhans (asterisk). H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eE. Higher magnification of Fig 3D. showing necrosis of islets cells as evidenced by nuclear pyknosis (arrow mark). H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eF. Strong immune staining of the non-structural protein of FMDV 3A in the Langerhans cells (asterisk). IHC.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/33193f75ce35b86d2f7644a6.png"},{"id":87401844,"identity":"c8cff4fc-f7bc-4fa3-9680-8fddb3323cf6","added_by":"auto","created_at":"2025-07-23 11:58:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5284698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental FMDV induced changes in the pancreas of a HF crossbred heifer (#A687) that succumbed on day 49 post-infection. \u003c/strong\u003eRepresentative photomicrographs of exocrine (A and B) and endocrine pancreas (C and D).\u003c/p\u003e\n\u003cp\u003eA. Regenerative changes in the pancreatic acinar cells (asterisk). H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eB. Mild to moderate cytoplasmic immunolabeling of non-structural protein of FMDV3A in the acinar (asterisk) and islets of Langerhans cells (arrow mark). IHC\u003c/p\u003e\n\u003cp\u003eC. Degenerative and necrotic changes in some of the islets of Langerhans (arrow mark) indicating persistence of lesions. H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eD. Note moderate cytoplasmic immunolabeling of the non-structural protein of FMDV 3A in the Langerhans cells (arrow mark). IHC.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/a98c4413a8ffc84b4b38b7a5.png"},{"id":88474369,"identity":"291b6e8f-d3b8-445c-a5bf-840d48a21368","added_by":"auto","created_at":"2025-08-06 20:31:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12848485,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/70be59f8-91e2-491a-883c-0290708a499a.pdf"},{"id":87403025,"identity":"5a1970c4-f8f2-42ce-a449-e6b93df96b55","added_by":"auto","created_at":"2025-07-23 12:14:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":992140,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7157311/v1/611c512913a408bce509ecba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Experimental Foot-and-mouth disease virus (FMDV) infection induces reversible exocrine pancreatic insufficiency and irreversible endocrine pancreatic insufficiency in Holstein-Friesian crossbred heifers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFoot-and-mouth disease (FMD) is one of the most highly contagious viral diseases affecting cloven-hoofed animals, including cattle, buffalo, swine, sheep, goats, bison, and deer worldwide. Due to its broad host range, rapid transmission, and significant economic impact, the OIE (2000) has classified FMD as the most dangerous disease affecting animals. The economic consequences of FMD vary by region; in enzootic areas, it causes major losses due to reduced productivity and trade restrictions, with estimated annual costs ranging from \u003cspan\u003e$\u003c/span\u003e6.5 to \u003cspan\u003e$\u003c/span\u003e21\u0026nbsp;billion (Alhaji et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In regions or countries free of FMD, outbreaks can lead to losses exceeding \u003cspan\u003e$\u003c/span\u003e1.5\u0026nbsp;billion annually (Knight-Jones and Rushton, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough most affected adult animals recover clinically within 2\u0026ndash;3 weeks of infection, they often do not regain their productivity and reproductive efficiency for a long period. In fact, 10\u0026ndash;20% of animals affected by FMD may permanently lose some of their productivity (Kitching, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Key concerns for animals recovering from FMD in enzootic areas include reduced milk production (Bayissa et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), poor fertility (Chaters et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), decreased draft capacity (Perry and Randolph, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and inadequate weight gain (Rufael et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The pancreas, an endocrine and exocrine gland, plays a vital role in digesting and metabolizing carbohydrates, fats, and proteins. In bovines, productive and reproductive functions depend on effective metabolism (Laskowski et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Metabolic challenges can lead to lower productivity, reproductive issues, and decreased fertility in ruminants (De Koster and Opsomer, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Laskowski et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe pancreas is known to serve as a preferred site for the replication of the foot-and-mouth disease virus (FMDV) in adult mice (Sanz-Ramos et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and as a site for persistence in infected cattle (Alexandersen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). FMDV in pancreatic tissue may damage the islets of Langerhans and pancreatic acinar cells, ultimately causing pancreatic dysfunction. Several studies have observed injury to pancreatic acinar cells and islets of Langerhans during the acute phase of FMD in cattle (Sutmoller et al., 2003; Ghanem and Abdel-Hamed, 2010; Sahoo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, research on the long-term effects of FMDV infection on the function of both the exocrine and endocrine pancreas is currently limited. Understanding these short-term and long-term effects is essential for developing therapies or remedial measures to mitigate production and reproduction losses in cattle. Therefore, this study was designed to evaluate the short-term and long-term effects of experimental FMDV infection on the endocrine and exocrine pancreatic functions in Holstein-Friesian crossbred heifers.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnimals\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFourteen apparently healthy Holstein Friesian (HF) crossbred heifers, aged 8 to 10 months, were selected for this study. None of these heifers had a history of previous FMDV infection, nor had been vaccinated against the virus. The heifers were dewormed and quarantined for a period of four weeks for acclimatization. They were screened for the presence of FMDV antibodies using an \u003cem\u003ein-house\u003c/em\u003e ELISA kit developed by ICAR-IVRI, Bengaluru, which detects FMDV non-structural proteins (NSP). Animals that did not show the presence of NSP antibodies were further examined for virus neutralization test (VNT). Only those animals that exhibited a virus neutralization (VN) titer of ≤8 (considered seronegative) were included in the study. The heifers were then randomly inoculated with FMDV serotype O by intradermolingual (n=7) or mock-infected with saline (n=7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experimental protocol received ethical approval from the Institutional Animal Ethics Committee of the ICAR - Indian Veterinary Research Institute in Bengaluru, as well as from the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under reference number V-11011(13)/13/2023-CPCSEA-DADF, dated January 5, 2024. The experiment was conducted in compliance with CPCSEA regulations.\u003c/p\u003e\n\u003cp\u003eExperimental infection of animals\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA bovine-derived FMDV serotype O/IND/R2/75 was used for experimental infection. Heifers were inoculated with 4.0 log\u003csub\u003e10\u0026nbsp;\u003c/sub\u003eBID\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e(50% bovine tongue infectious dose) of virus suspended in 0.2 mL of media via intradermolingual route. 0.2 mL of sterile normal saline was injected by intradermolingual route into mock-infected animals. Both FMDV-infected and mock-infected animals were housed separately in the biocontainment facility under iso-managerial conditions. Clinical observations were recorded from 2 days before the experimental infection to 180 days post-infection (dpi).\u003c/p\u003e\n\u003cp\u003eConfirmation of FMDV infection and serotype\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConfirmation of foot-and-mouth disease virus (FMDV) infection in the animals was performed using an \u003cem\u003ein-house\u003c/em\u003e serotype-differentiating antigen detection ELISA kit developed by the Directorate of Foot-and-Mouth Disease in India, as outlined by Bhattacharya et al. (1996). The serotype-differentiating antigen detection ELISA performed using samples from the epithelium of the tongue or the interdigital space confirmed that the infection was caused by serotype O of the FMD virus (FMDV).\u003c/p\u003e\n\u003cp\u003eSample collection\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBlood samples were collected on the day of infection (day 0) and at various time points following the experimental infection, namely, 7, 14, 30, 60, 90, 120, 150, and 180 dpi. Approximately 10 mL of blood was drawn from the jugular vein of each animal using a vacutainer coated with heparin (BD, Franklin, USA) as an anticoagulant for plasma samples. Additionally, another 10 mL of blood was drawn into vacutainers containing a clot activator (BD, Franklin, USA) for serum collection. Each vial was labelled with the animal number, date, and time of collection. The tubes were centrifuged at 3000g for 20 min to clarify the samples. Following centrifugation, the serum and plasma were transferred to fresh 4 mL Eppendorf tubes and stored at -20°C for further analysis.\u003c/p\u003e\n\u003cp\u003eBiochemical analysis\u003c/p\u003e\n\u003cp\u003ePlasma glucose, serum amylase, lipase, triglycerides, and cholesterol were estimated by colorimetric method using commercial kits (GenX, Proton Biologicals, India) and a Photometer version NP80 (Sl No. M81334; Implen GmbH, Germany). Serum insulin was estimated using bovine enzyme immunoassay (EIA; Mercodia, Winston Salem, NC; with the sensitivity of ≤ 0.025 µg/L). Plasma NEFA concentration was estimated by colorimetric method (Clementia Biotech, New Delhi, India, Sensitivity: 0.05 mmol/L, Detection range: 0.05-2.0 mmol/L; Inter assay CV: 1.55%, Intraassay CV: 0.6%). Plasma BHB concentration was estimated by colorimetric method (Abcam, ab83390, Sensitivity: 0.01 mM, Range: 0.01 mM - 0.2 mM, Inter assay CV: 1.55%, Intraassay CV: 0.6%). Trypsin-like immunoreactivity (TLI) was estimated by Canine specific TLI (Trypsin-like immunoreactive protein) ELISA kit (Catalog # ELK 0307, ELK Biotechnology Co. Ltd., India). The analytical sensitivity of TLI kit was 11.3 ng/ml with a detection limit of 31.25-2000 ng/ml. The manufacturer reported intra-assay and inter-assay CV (=SD/mean×100) were \u0026lt;8% and \u0026lt;10% respectively. Glycated haemoglobin was estimated by ion exchange chromatography method (Coral clinical systems, India).\u003c/p\u003e\n\u003cp\u003eHistopathology and immuno-histochemistry\u003c/p\u003e\n\u003cp\u003eOut of seven experimentally infected HF crossbred heifers, one heifer died on dpi 12, and another died on dpi 49. A necropsy examination was conducted, and pancreatic tissues were collected in 10% neutral buffered formalin for histopathology and immunohistochemistry following standard guidelines (Luna, 1968; Ramos-Vara, 2017).\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eA two-way repeated measures ANOVA was conducted to evaluate the time-dependent effects of FMDV infection on the endocrine pancreas, exocrine pancreas, and energy metabolism at various days post-infection. A Bonferroni test was applied to compare the pairwise mean differences between different days of FMDV infection. A significance level was set at 5%. Data analysis and chart preparation were performed using GraphPad Prism version 9.5.4.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOnset of hyperpnea and pyrexia was recorded by 48 h post-inoculation of FMDV; excessive salivation, vesicular lesions in the oral cavity, and nasal discharge appeared by 72 h post-inoculation. Vesicular lesions at the interdigital space were recorded by 96 h post-inoculation (Supplementary figure 1). Oral lesions were the first to heal, while hoof lesions healed by two weeks post-inoculation. After the apparent clinical convalescence by 3 - 4 weeks from the acute lesion, FMDV infected heifers developed ruffled hair coat with hypopigmented hairs in the trunk area around 5-6 week (3/7) and cracked hooves around 6–7-week post-inoculation (4/7). Onset of panting was consistently observed around 8 - 9 weeks post-inoculation in all the FMDV-infected heifers (7/7) (Supplementary Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring the acute phase, a significant increase in the concentrations of serum amylase, lipase, and TLI was observed on dpi 7 (Figure 1A, 1C, and 1E) in FMDV-infected heifers compared to mock-infected heifers. Elevated concentrations of amylase and TLI returned to pre-infection concentrations by dpi 60 (Figure 1D and 1F), while that of lipase was on dpi 120. Changes in the serum amylase and lipase indicated their potential utility as a biomarker during the first 60 days of FMD infection. In striking contrast to exocrine pancreatic enzymes, serum concentrations of insulin showed a significant decrease by dpi 14 (Figure 1G) in FMDV-infected heifers that persisted till dpi 180 (Figure 1H).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Among the indicators of energy metabolism, serum triglycerides, plasma non-esterified fatty acids (NEFA), and plasma β-hydroxybutyrate showed a significant increase by dpi 14 (P\u0026lt;0.01; Figure 2E, 2G, and 2I) in the FMDV infected heifers and remained elevated till dpi 180 (P\u0026lt;0.0001; Figure 2F, 2H, and 2J). However, the concentrations of plasma glucose and glycated Hb (%) were comparable till dpi 14 (P\u0026gt;0.05; Figure 2A and 2C) between the FMDV-infected heifers and mock-infected group. Onset of hyperglycemia in the FMD-infected heifers was apparent by dpi 30 while, glycated Hb (%) showed a significant rise by dpi 90 (Fig. 2D). Similar to lipids, plasma glucose and glycated Hb (%) remained elevated till dpi 180 in the heifers (P\u0026lt;0.0001; 2B and 2D). The results are further supported by the histopathological and immunohistochemical findings in the pancreas of heifers that died on dpi 12 (Heifer #A681) and dpi 49 (Heifer #A687). Histopathology of the pancreas revealed necrosis and mononuclear infiltration of the acinar cells (Fig. 3 A and B) and Langerhans (Fig. 3 D and E) in the heifer that succumbed on dpi 12. Intense immunolocalization of the non-structural protein 3A of FMDV suggested colonization of FMD and possible viral replication in the exocrine (Fig. 3C) and endocrine (Fig. 3F) pancreas. In contrast to the regenerative changes in the acinar cells (Fig. 4A), degenerative and necrotic changes persisted in the Islet cells of the pancreas (Fig. 4C) in the heifer that died on dpi 49. Though moderate immunostaining of the non-structural protein 3A of FMDV indicated persistence of FMDV in the exocrine and endocrine pancreas till dpi 49 (Fig. 4 B and D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe clinical signs observed during both the acute and chronic phases of FMDV infection in the HF crossbred heifers align with previous findings (Ghanem and Ahmed, 2010; Kamal et al., 2018). Symptoms such as fever, excessive salivation, rapid breathing, vesicular and ulcerative lesions in the oral cavity and interdigital space can be linked to the viraemic phase, during which FMDV replicates in the oropharyngeal epithelium and spreads systemically to other tissues. This replication causes vesicular and ulcerated lesions in both the mouth and interdigital space of the feet (Sutmoller et al., 2003). Chronic signs like panting, rough hair coat, lameness, and overgrown hooves are consistent with previous reports in Egyptian cattle, where these symptoms recorded three months after an FMD outbreak (Ghanem and Ahmed, 2010). Panting and rough hair coat observed could be imputed to the alterations in the metabolic functions (Ghanem and Ahmed, 2010). Chronic lameness might lead to improper wear of the hooves, resulting in overgrown hooves (Belsham et al., 2024). Chronic signs such as cracked hooves with partial loss of black pigmentation in the hair coat have not been reported elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExocrine pancreatic function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe recorded the trend of circulating concentrations of serum amylase, lipase, and TLI from dpi 0 to 180 day post-FMDV infection to assess the temporal changes in exocrine pancreatic damage, which is often challenging to diagnose in ruminants (Vargas-Rodriguez et al., 2014; Guo et al., 2021). Since amylase is also produced by rumen microflora (Vargas-Rodriguez et al., 2014), and salivary glands contribute to lipase, a significant increase in two or more digestive enzymes such as amylase, lipase, and TLI indicates pancreatic injury or inflammation in ruminants. In cows infected with FMDV, serum levels of amylase and lipase increased significantly during dpi 7-30 and dpi 7-90, respectively (Fig. 1 A to D). Previous studies have also reported elevated levels of amylase and lipase in cattle infected with FMDV (Kamal et al., 2018; Soltani et al., 2020). Additionally, a notable rise in serum TLI levels was observed between dpi 7-14 in FMDV-infected cows, suggesting damage to the pancreatic acini (Flamion et al., 1987). Trypsin is released into circulation when these acinar cells are damaged, leading to increased TLI levels. This study is the first to report elevated TLI in FMDV-infected cattle, though it is a diagnostic marker for acute pancreatitis in humans, dogs, and cats (Flamion et al., 1987; Xenoulis, 2015).\u003c/p\u003e\n\u003cp\u003eElevated amylase, lipase, and TLI in FMDV-infected HF crossbred heifers may be due to damage caused by FMDV infection in exocrine pancreatic acinar cells. This is supported by previous studies that have extensively documented degeneration and necrotic changes in the pancreatic acinar cells during FMDV infection in cattle (Yeotikar et al., 2003; Ghanem and Abdel-Hamid, 2010; Nahed et al., 2010; Artz et al., 2011; Sahoo et al., 2023). Similar pathological findings were observed in the exocrine pancreas of an HF crossbred heifer that succumbed to experimental FMDV infection on dpi 12 in the present study (Figure 3A). Intense immunostaining of the 3A protein of FMDV on dpi 12 (Fig. 3C) in the pancreatic acinar cells (Heifer #A681) indicated a high viral load. It is reported that FMDV has a special affinity for pancreatic tissue and can replicate there, leading to inflammatory changes in the pancreas (Alexandersen et al., 2002; Sutmoller et al., 2003; Sanz-Ramos et al., 2008). The serum concentrations of exocrine pancreatic enzymes returned to pre-infection levels by dpi 60 to 90, indicating resolution of inflammation. This is supported by the regenerative changes observed in the pancreatic acinar cells of the HF crossbred heifer that succumbed on dpi 49 (Fig. 4A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndocrine pancreatic function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental infection of the heifers with FMDV resulted in ~20% reduction in the serum insulin concentrations by dpi 14 that further decreased to ~40% by dpi 30 dpi compared to mock-infected heifers (Fig. 1 G and H). Seminal experiment by Barbni et al. (1966) demonstrated induction of diabetes mellitus in the FMD infected cattle. Hypoinsulinemia was reported in cattle after recovery from natural FMD infection (Nahed, 2010). Our unpublished observations indicate insulin insufficiency in the purebred HF and Jersey cows till day 180 post-FMD outbreak. A significant reduction in serum insulin concentrations in FMDV-infected HF crossbred heifers might be due to virus-induced destruction of insulin-secreting β-cells in the pancreas as evidenced by the presence of FMDV antigens in the endocrine pancreas and absence of regenerative changes in the endocrine pancreas of a heifer that died on day 49 post-infection (Fig. 4 C and D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to the bovine viral diarrhea virus, FMDV has also been associated with the development of type 1 diabetes in cattle (Nahed, 2010). FMDV can directly damage insulin-producing pancreatic β-cells through viral replication (Barbni et al., 1966; Sanz-Ramos et al., 2008) or through autoimmune reactions triggered by the virus. However, none of the experimentally infected HF crossbred heifers in this study developed glycosuria, indicating insulin insufficiency rather than insulin deficiency. In contrast to the resolution of exocrine pancreatic damage around dpi 120 as evidenced by normal concentrations of pancreatic enzymes (Fig. 1 B, D, and F), decreased insulin concentration persisted till dpi 180 (Fig. 1H) in the FMDV-infected HF crossbred heifers. Presence of FMDV antigens and absence of regenerative changes in the endocrine pancreas by dpi 49 in a heifer #A687) also support the insulin insufficiency till dpi 180. Collectively, the exocrine pancreas function was restored by dpi 90, while endocrine dysfunction persisted until dpi 180.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnergy metabolism\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA significant increase in the plasma glucose by dpi 30 in FMDV-infected HF crossbred heifers (Fig. 2B; P \u0026lt; 0.0001) might be due to insulin insufficiency. Hyperglycemia is a consistent finding in cattle (Yeotikar et al., 2003; Gokce et al., 2004; Bakrakati et al., 2015; Kamal et al., 2018; Soltani et al., 2020) and sheep (Gattani et al., 2011) recovered from FMD. Glycated hemoglobin, a fraction of hemoglobin that binds to plasma glucose, serves as a marker for evaluating glucose levels over an extended period (Neumann, 2020; Norris and Schermerhorn, 2022). A significant increase (P \u0026lt; 0.0001) in glycated hemoglobin levels observed in FMDV-infected HF crossbred heifers from 90 to 180 dpi, can be attributed to the chronic hyperglycemia present in these animals.\u0026nbsp;Additionally, hypertriglyceridemia from dpi 14 to 180 in the FMDV-infected heifers than in\u0026nbsp;mock-infected heifers\u0026nbsp;(Fig. 2 E and F; P \u0026lt; 0.0001), indicates impaired insulin action.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsulin typically activates lipoprotein lipase to liberate fatty acids from the triglycerides. Therefore, FMDV-induced insulin insufficiency might have disrupted the activation of lipoprotein lipase, resulting in the accumulation of triglycerides. Our findings are in agreement with Soltani et al. (2020), who also reported hypertriglyceridemia in animals affected by FMD. A significant increase in the plasma concentrations of NEFA and BHB from dpi 14 to 180\u0026nbsp;(Fig. 2 I and J; P=0.0004)\u0026nbsp;may be linked to hypoinsulinemia in the FMDV-infected heifers.\u0026nbsp;During hypoinsulinemia, the body mobilizes subcutaneous fat, leading to the production of NEFA (Adewuyi et al., 2005). Hypoinsulinemia also promotes the entry of NEFA into mitochondria, which in turn facilitates the formation of ketone bodies such as BHB. Our findings are consistent with earlier reports (Kamal et al., 2018) that indicated elevated levels of NEFA and BHB in animals affected by FMDV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and immunohistochemistry of the pancreas\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe major histopathological findings observed in the HF crossbred heifer that died on the 12\u003csup\u003eth\u003c/sup\u003e day post-FMDV included diffuse necrosis of the acinar cells, along with the infiltration of mononuclear cells such as lymphocytes, macrophages, and plasma cells in the exocrine pancreas. Additionally, degeneration and necrosis of the islets of Langerhans in the endocrine pancreas were noted. Similar pancreatic pathology has been documented in previous studies involving FMD-affected calves (Sahoo et al., 2023), cattle (Barker et al., 1992), and gazelles (Berkowitz et al., 2010). These findings are supported by elevated concentrations of circulating pancreatic enzymes, including amylase, lipase, and TLI, observed in FMD-infected HF crossbred heifers from dpi 7 to 120 (Fig. 1 A to F). Elevated pancreatic enzymes are also reported during the acute phase of FMD (Kamal et al., 2018; Soltani et al., 2020). Intense cytoplasmic immunolabeling for FMDV antigen in the islets of Langerhans indicated colonization and possible replication of the virus in these areas. The pancreas is known to be the preferred site for viral replication in adult mice, with the highest viral load detected 24 h \u0026nbsp;post-FMDV infection (Sanz-Ramos et al., 2008). Similar microscopic lesions, such as lymphocytic infiltration in both pancreatic acinar cells and islets of Langerhans, necrosis of acinar cells and islets of Langerhans were noticed in the mice infected with FMDV (Sanz-Ramos et al., 2008). Several reports suggest that FMDV can replicate in the pancreatic tissue of cattle, resulting in necrosis and degeneration of both the endocrine and exocrine pancreas (Alexandersen et al., 2002; Kamal et al., 2018).\u003c/p\u003e\n\u003cp\u003eIn contrast, histopathological examination of the pancreatic tissue from another FMDV-infected HF crossbred heifer that died on dpi 49 revealed regenerative changes in the exocrine pancreas. These observations align with the normal levels of pancreatic enzymes, such as amylase, lipase, and TLI, found in FMDV-infected HF crossbred heifers by 120 dpi in the current study. However, necrotic changes in the islets of Langerhans persisted in the endocrine pancreas, which corresponds with persistently decreased concentrations of serum insulin in experimentally infected HF crossbred heifers. Mild to moderate cytoplasmic immunolabeling for FMDV antigen was noted in both acinar cells and the islets of Langerhans, indicating the persistence of FMDV in the pancreas. This notion is supported by Alexandersen et al. (2002), who suggested that FMDV can persist in the pancreas along with other organs such as the mammary gland, testicles, pituitary gland, and thyroid for extended periods. Furthermore, Arzt et al. (2011) indicated that disruption of pancreatic function accounts for the hyperglycemia observed in cattle that have recovered from FMD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eExperimental FMDV infection induced reversible damage to exocrine pancreas while irreversible damage to the endocrine pancreas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Director and Joint Director of ICAR – Indian Veterinary Research Institute, Regional Campus, Bengaluru for the necessary support. The authors thank the Department of Biotechnology, Ministry of Science and Technology and Government of India for the financial support received for this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was funded by\u0026nbsp;Department of Biotechnology,\u0026nbsp;Ministry of Science and Technology, Government of India to Dr. Priyanka Mahadappa vide\u0026nbsp;grant No.BT/PR30711/BIC/101/1135/2018.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePriyanka Mahadappa conceptualized the study.All authors contributed to the study design. Material preparation, data collection and analysis were performed by Padmanaban Udhayabanu, Priyanka Mahadappa, Karikalan Mathesh, Saravanan Paramasivam, Aadhithya Muthuswamy, Umapathi V, B H Manjunatha Patel, Veerakyathappa Bhanuprakash, Dechamma Hosuru Joyappa, Pallab Chaudhuri, and Narayanan Krishnaswamy. The first draft of the manuscript was written by Priyanka Mahadappa, Narayanan Krishnaswamy, and Padmanaban Udhayabanu. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdewuyi AA, Gruys E, van Eerdenburg FJCM (2005) Non esterified fatty acids (NEFA) in dairy cattle. A review. Vet Q 27:117\u0026ndash;126\u003c/li\u003e\n\u003cli\u003eAlexandersen S, Zhang Z, Donaldson AI (2002) Aspects of the persistence of foot-and-mouth disease virus in animals\u0026mdash;the carrier problem. Microbes Infect 4:1099\u0026ndash;1110. https://doi.org/https://doi.org/10.1016/S1286-4579(02)01634-9\u003c/li\u003e\n\u003cli\u003eAlhaji NB, Amin J, Aliyu MB, et al (2020) Economic impact assessment of foot-and-mouth disease burden and control in pastoral local dairy cattle production systems in Northern Nigeria: A cross-sectional survey. Prev Vet Med 177:104974\u003c/li\u003e\n\u003cli\u003eArzt J, Juleff N, Zhang Z, Rodriguez LL (2011) The pathogenesis of foot-and-mouth disease I: viral pathways in cattle. Transbound Emerg Dis 58:291\u0026ndash;304\u003c/li\u003e\n\u003cli\u003eBarboni E, Manocchio I, Asdrubali G (1966) Osservazioni sul diabete dei bovini da afta epizootica sperimentale. Nuovi Ann Ig Microbiol 17:223\u0026ndash;226\u003c/li\u003e\n\u003cli\u003eBarkakati J, Sarma S, Kalita D (2015) Effect of foot and mouth disease on haematological and biochemical profile of cattle. Indian J Anim Res 49:. https://doi.org/10.18805/ijar.5588\u003c/li\u003e\n\u003cli\u003eBarker IK, Van Dreumel AA, Palmer N (1993) The Alimentary System. In: Jubb KF, Kennedy PC, Palmer N (eds) Pathology of Domestic Animals. Academic Press Inc, pp 13\u0026ndash;18\u003c/li\u003e\n\u003cli\u003eBayissa B, Ayelet G, Kyule M, et al (2011) Study on seroprevalence, risk factors, and economic impact of foot-and-mouth disease in Borena pastoral and agro-pastoral system, southern Ethiopia. Trop Anim Health Prod 43:759\u0026ndash;766. https://doi.org/10.1007/s11250-010-9728-6\u003c/li\u003e\n\u003cli\u003eBelsham GJ, B\u0026oslash;tner AG, Lohse L (2021) Foot-and-mouth disease in animals. In: MSD Manual-Veterinary Manual\u003c/li\u003e\n\u003cli\u003eBerkowitz A, Waner T, King R, et al (2010) Description of the pathology of a gazelle that died during a major outbreak of foot-and-mouth disease in Israel : clinical communication. J S Afr Vet Assoc 81:62\u0026ndash;64\u003c/li\u003e\n\u003cli\u003eBhattacharya S, Pattnaik B, Venkataramanan R (1996) Development and application of sandwich enzyme-linked immunosorbent assay (ELISA) for type identification of foot-and-mouth disease (FMD) virus in direct field materials. Indian J Anim Sci 66:1\u0026ndash;9\u003c/li\u003e\n\u003cli\u003eChaters G, Rushton J, Dulu TD, Lyons NA (2018) Impact of foot-and-mouth disease on fertility performance in a large dairy herd in Kenya. Prev Vet Med 159:57\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eDe Koster JD, Opsomer G (2013) Insulin Resistance in Dairy Cows. Veterinary Clinics of North America: Food Animal Practice 29:299\u0026ndash;322. https://doi.org/https://doi.org/10.1016/j.cvfa.2013.04.002\u003c/li\u003e\n\u003cli\u003eFlamion B, Delhaye M, Horanyi Z, et al (1987) Comparison of elastase-1 with amylase, lipase, and trypsin-like immunoreactivity in the diagnosis of acute pancreatitis. Am J Gastroenterol 82:532\u0026ndash;535\u003c/li\u003e\n\u003cli\u003eGattani A, Gupta K, Joshi G, Gupta S (2011) Metabolic profile of foot and mouth disease stressed sheep in semi arid region. Journal of Stress Physiology \u0026amp; Biochemistry 7:148\u0026ndash;153\u003c/li\u003e\n\u003cli\u003eGhanem MM, Abdel-Hamid OM (2010) Clinical, haematological and biochemical alterations in heat intolerance (panting) syndrome in Egyptian cattle following natural foot-and-mouth disease (FMD). Trop Anim Health Prod 42:1167\u0026ndash;1173\u003c/li\u003e\n\u003cli\u003eG\u0026ouml;k\u0026ccedil;e G, G\u0026ouml;kce Hali̇l \\.Ibrahi̇m and G\u0026uuml;neş V, Erdoğan HM, \u0026Ccedil;i̇ti̇l M (2004) Alterations in some haematological and biochemical parameters in cattle suffering from foot- and -mouth disease. Turk J Vet Anim Sci 28:723\u0026ndash;727\u003c/li\u003e\n\u003cli\u003eGuo L, Yao J, Cao Y (2021) Regulation of pancreatic exocrine in ruminants and the related mechanism: The signal transduction and more. Animal Nutrition 7:1145\u0026ndash;1151.\u003c/li\u003e\n\u003cli\u003eKamal E, Salama M, Heakal NA (2018) ALTERATIONS IN SOME BIOCHEMICAL PARAMETERS IN CATTLE AFFECTED WITH FOOT AND MOUTH DISEASE IN DAKAHLIA GOVERNORATE, EGYPT. Mansoura Veterinary Medical Journal\u003c/li\u003e\n\u003cli\u003eKitching RP (2002) Clinical variation in foot and mouth disease: cattle. Rev Sci Tech 21:499\u0026ndash;504\u003c/li\u003e\n\u003cli\u003eKnight-Jones TJD, Rushton J (2013) The economic impacts of foot and mouth disease - What are they, how big are they and where do they occur? Prev Vet Med 112:161\u0026ndash;173\u003c/li\u003e\n\u003cli\u003eLaskowski D, Sjunnesson Y, Humblot P, et al (2016) The functional role of insulin in fertility and embryonic development-What can we learn from the bovine model? Theriogenology 86:457\u0026ndash;464\u003c/li\u003e\n\u003cli\u003eLuna LG (1968) Manual of histologic staining methods of the Armed Forces Institute of Pathology. McGraw-Hill, New York\u003c/li\u003e\n\u003cli\u003eNahed ST (2010) Investigation of serum insulin and cortisol concentrations in foot and mouth disease-infected cattle in relation to changes in serum biochemical variables and protein electrophoretic fractionation profile.\u003c/li\u003e\n\u003cli\u003eNeumann S (2020) Reference interval of hemoglobin A1c and influence of hematological parameters on its serum concentration in dogs. Vet Med Int 2020:7150901\u003c/li\u003e\n\u003cli\u003eNorris O, Schermerhorn T (2022) Relationship between HbA1c, fructosamine and clinical assessment of glycemic control in dogs. PLoS One 17:e0264275\u003c/li\u003e\n\u003cli\u003ePerry BD, Randolph TF (2003) The economics of foot and mouth disease, its control and its eradication. Elsevier SAS, Paris\u003c/li\u003e\n\u003cli\u003eRamos-Vara JA (2017) Principles and methods of immunohistochemistry. In: Methods in Molecular Biology. Springer New York, New York, NY, pp 115\u0026ndash;128\u003c/li\u003e\n\u003cli\u003eRufael T, Catley A, Bogale A, et al (2008) Foot and mouth disease in the Borana pastoral system, southern Ethiopia and implications for livelihoods and international trade. Trop Anim Health Prod 40:29\u0026ndash;38. Sahoo M, Singh R, Kumar P, et al (2023) Novel pathologic findings and viral antigen distribution in cattle and buffalo calves naturally infected with Foot-and-Mouth disease virus. Veterinary Quarterly 43:1\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eSanz-Ramos M, D\\\u0026rsquo;\\iaz-San Segundo F, Escarm\\\u0026rsquo;\\is C, et al (2008) Hidden virulence determinants in a viral quasispecies in vivo. J Virol 82:10465\u0026ndash;10476\u003c/li\u003e\n\u003cli\u003eSoltani H, Aslani M reza, Mohebbi A, Mokhtari A (2020) Serum biochemical and oxidative status in Holstein cattle affected with foot and mouth disease. Iran J Vet Sci Technol 12:19\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eSutmoller P, Barteling SS, Olascoaga Raul Casas and Sumption KJ (2003) Control and eradication of foot-and-mouth disease. Virus Res 91:101\u0026ndash;144\u003c/li\u003e\n\u003cli\u003eSutmoller P, Casas OR (2002) Unapparent foot and mouth disease infection (sub-clinical infections and carriers): implications for control. Rev Sci Tech 21:519\u0026ndash;529\u003c/li\u003e\n\u003cli\u003eVargas-Rodriguez CF, Engstrom M, Azem E, Bradford BJ (2014) Effects of dietary amylase and sucrose on productivity of cows fed low-starch diets. J Dairy Sci 97:4464\u0026ndash;4470.\u003c/li\u003e\n\u003cli\u003eWOAH (2024) WOAH Terrestrial Manual 2022. In: The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual), 13th edn.\u003c/li\u003e\n\u003cli\u003eXenoulis PG (2015) Diagnosis of pancreatitis in dogs and cats. Journal of Small Animal Practice 56:13\u0026ndash;26. https://doi.org/https://doi.org/10.1111/jsap.12274\u003c/li\u003e\n\u003cli\u003eYeotikar P V, Bapat ST, Bilolikar SC, Kulkarni SS (2003) Metabolic profile of healthy cattle and caftle affected by foot‐and‐mouth disease. Vet Rec 153:19\u0026ndash;20\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":"Amylase, Foot-and-mouth Disease, Insulin, Lipase, Trypsin-like immunoreactivity","lastPublishedDoi":"10.21203/rs.3.rs-7157311/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7157311/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs hyperglycemia is a consistent finding in cattle recovered from FMD, we hypothesized that damage to the pancreas by FMD virus (FMDV) is the basis for the alterations in energy metabolism. Accordingly, crossbred heifers were either inoculated with virulent FMDV serotype O by intradermolingual (n\u0026thinsp;=\u0026thinsp;7) or mock-infected with saline (n\u0026thinsp;=\u0026thinsp;7). Blood samples were collected on day 0, 7, 14, 30, 60, 90, 120, 150, and 180 post-infection (dpi) to determine the concentrations of exocrine pancreatic enzymes, insulin, and indicators of energy metabolism. The results indicated a significant elevation in the serum amylase, lipase, and TLI by dpi 7 in FMDV-infected heifers that returned to baseline by dpi 60\u0026ndash;120. In contrast, decrease in the serum insulin, first recorded on dpi 14, persisted till dpi 180 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The circulating concentrations of glucose, glycated hemoglobin, triglycerides, NEFA and BHB showed a significant increase by dpi 30 and did not recede to basal level till dpi 180 in the FMDV-infected group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Pancreatic histopathology and immunohistochemistry in a heifer that died on 12 dpi revealed necrotic and inflammatory changes in the pancreas with intense immunolabeling for FMDV antigens both in the pancreatic acini and islets. In another heifer that died on dpi 49, regenerative changes were found in the exocrine pancreas, while necrotic changes persisted in the islets of Langerhans, with moderate FMDV antigen labeling. It was concluded that experimental FMDV infection-induced endocrine pancreatic insufficiency was irreversible, whereas exocrine pancreatic function could be restored by dpi 120.\u003c/p\u003e","manuscriptTitle":"Experimental Foot-and-mouth disease virus (FMDV) infection induces reversible exocrine pancreatic insufficiency and irreversible endocrine pancreatic insufficiency in Holstein-Friesian crossbred heifers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 11:58:45","doi":"10.21203/rs.3.rs-7157311/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":"44506af7-66fc-4d2c-af43-b66779f13643","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-06T20:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 11:58:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7157311","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7157311","identity":"rs-7157311","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}