Novel pathologic findings and viral antigen distribution in cattle and buffalo calves naturally infected with Foot-and-Mouth disease virus

Abstract

The Foot-and-Mouth disease is highly contagious acute viral disease of livestock inflicting huge economic loss to the farmers. The limited knowledge regarding the pathological lesions vis-a-vis distribution of the FMDV in lesser explored endocrine glands and important vital organs other than the target organs of infected calves prompted us to take the present investigation to have detailed insight into the pathogenesis. The systematic necropsy of 37 dead calves (cattle-28 and buffalo-9) was conducted, and thin representative tissue pieces from the affected organs were collected in 10% neutral buffered formalin (NBF) for pathological and immunohistochemical investigations. The genomic detection and its serotyping were done by RT-PCR and multiplex-PCR, respectively. Necropsy examination in all cases showed myocardial lesions resembling ‘tigroid heart appearance’. Other organ specific lesions include vesiculo-ulcerative stomatitis, edema of the lungs, petechial hemorrhages, edema of the endocrines, and gastroenteritis. Histopathological examination showed varying sizes of vesicles and ulcerations in stratified squamous epithelium of the tongue, acute necrotizing myocarditis, lymphoid depletion in lymphoid tissues, hepatitis, pancreatitis, thymic hyperplasia, thyroiditis, adrenitis, and enteritis. Positive immunolabeling for viral antigens was observed in endocrine glands, lymphoid organs, lungs, liver, kidneys, and intestine, in addition to other typical locations. The thyroid, adrenal glands, and pancreas, in addition to the tongue and heart, are the tissue of choice for sampling in the field during epidemics. Further, the viral genome and serotype A was confirmed in the affected tissues. This study provides insights into novel tissue tropism and pathogenesis in young calves naturally infected with FMDV.

Keywords:

• Calves
• foot-and-mouth disease
• immunohisto­chemistry
• multiplex PCR
• pathology
• RT-PCR
• serotype A

1. Introduction

Foot-and-mouth disease (FMD) is a highly contagious and one of the transboundary animal diseases (TADs) of cloven-footed animals, causing serious morbidity and mortality threats to livestock animals (Subramaniam et al. 2022). It is caused by pathogen of the genus Aphthovirus, which is part of the family Picornaviridae. FMD ranks first among the ‘Risk Group 4’ animal pathogens (Biswal et al. 2012), and is known for causing a serious threat to global food security, economic losses, reproductive performance, and animal trade (Ranjan et al. 2016; Govindaraj et al. 2021). Of its 7 serotypes, serotype ‘O’ is widely prevalent in India (80–92%), followed by Asia1 (3–10%) and serotype A (3–8%) (Rudreshappa et al. 2012). The serotype ‘A’ virus is genetically more divergent when compared to the other two serotypes. The disease is clinically characterized by severe emaciation, high fever, lameness, and vesicular lesions on the mouth, tongue, feet, snout, and teats of infected animals. The mortality rates in adult animals are usually low-to-negligible. In contrast, high mortality is observed in suckling and young animals that present with few or no vesicular lesions. In these cases, death results from myocarditis in young calves up to 3 months old (Stenfeldt et al. 2014; Aktas et al. 2015; Ranjan et al. 2016). Earlier investigations suggested that mortality in young suckling ruminants and pigs varied from 20–75% in the most extreme cases, and it is highly age-dependent (Ryan et al. 2008; Aktas et al. 2015; Stenfeldt et al. 2016). Multiple studies conducted on specimens obtained during field outbreaks (Gulbahar et al. 2007; Aktas et al. 2015; Ranjan et al. 2016) and experimental samples (Gulbahar et al. 2011; Stenfeldt et al. 2014) have confirmed a relationship between acute foot-and-mouth disease virus (FMDV) infections and fatal myocarditis in young ruminants and pigs. Besides cardiac dysfunction, the clinically affected calves also exhibit a diverse array of clinical signs, indicating their tropism in various organs besides the target organ. Besides the tongue and heart, the tropism of FMDV in atypical organs has not been fully investigated. Although the clinical signs (panting, salivation, enteritis) exhibited by the affected calves presumed to be the dysfunction of various vital organs including the endocrine glands, the detailed investigation regarding the histopathological alterations in FMDV antigens colocalized sites in various organs, besides the target one, is not available.

Over the past three decades, multiple studies of FMDV have been performed in experimental settings (Pacheco et al. 2015; Stenfeldt et al. 2015) to study the events associated with the pathology and pathogenesis in adult animals. However, the organ tissue changes in relation to the distribution of FMDV antigens in young calves and subsequent clinical expressions of the disease have not been investigated during epidemics. It was hypothesized that the sites of viral replication in younger animals would be different than in adults, which underpins the site-specific pathological lesions leading to high mortality. Numerous old reports have shown the localization of the virus within the tongue and heart tissues of the affected animals using in-situ hybridization (ISH) (Brown et al. 1996; Durand et al. 2008; Zhang et al. 2016), confocal microscopy (Monaghan et al. 2005; O’Donnell et al. 2009), and fluorescent antibody techniques (Pacheco et al. 2015; Stenfeldt et al. 2015; Ranjan et al. 2016). However, a paucity of literature is available regarding the distribution of viral antigens, and studies have only been performed in a limited number of organs (Ryan et al. 2008; Arzt et al. 2009; Gulbahar et al. 2011) using immunohistochemistry (IHC). Over the past decade, the IHC technique has proved useful for identifying viruses and cell types involved in these diseases. Moreover, the technique is simple, cost-effective, and applicable to formalin-fixed paraffin-embedded tissue sections, yielding reliable results. In addition, we made an interesting observation regarding the pathological lesions and distribution of viral antigens of FMDV that mimic Enteroviruses. As part of the Picornaviridae family, Enteroviruses are known to be associated with hand-foot-and-mouth disease (HFMD) (Liu et al. 2013; Koh et al. 2016), myocarditis (Akuzawa et al. 2014; Muehlenbachs et al. 2015), pancreatitis (Mena et al. 2000; Chrysos et al. 2004; Zhang et al. 2016), and meningoencephalitis (Yu et al. 2014; Xing et al. 2016) in humans.

Little is known about the relationship between the pathology of FMDV and that of Enteroviruses with respect to lesions in the tongue, heart, pancreas, adrenal glands, and brain. We hypothesize that significant similarity exists between these two viruses with regard to their mechanisms of action and their host responses. Thus, the current study aimed to produce detailed insights into the gross, histopathological, and tissue-specific localization of FMDV in acutely infected calves under natural conditions.

2. Materials and methods

2.1. Study design

The tissues for this study were collected from 37 dead calves (28 bovine; 9 bubaline), which were submitted to the Postmortem Facility for necropsy, patho-anatomy, histopathology, immunohistochemical, and molecular analyses. These calves were brought dead from an organized dairy herd situated at Bareilly, consisting of 590 cattle and 198 buffaloes. The herd had a routine practice of twice-yearly vaccinations using the commercially available inactivated FMDV-trivalent vaccine (serotypes A, O, and Asia1). Out of a total of 788 vaccinated animals, 127 were infected between the ages of 2 months to 1 year, presenting with clinical signs suggestive of FMD. The majority of the affected animals (59 cases) were less than 4 months of age belonging to the Vrindavani breed, which is a synthetic crossbred strain of cattle containing exotic inheritance of Holstein-Friesian, Brown Swiss, Jersey cattle, and Hariana cattle breeds. Although both calves and adults were affected, mortality occurred only in calves. During the course of the disease, a total of 37 calves [females-20; cattle-16, buffalo-4 and males-17; cattle-12, buffaloe-5] of 127 affected animals died over a period of 1 month. The clinical evaluation and detection of FMD in live animals was done by the Scientists from Centre for Animal Disease Research and Diagnosis (CADRAD), IVRI. Further, the representative tissue samples of tongue and heart were sent to Directorate of Foot-and-Mouth Disease laboratory (DFMD, Mukteswar, India, and FAO Reference Laboratory for FMD for South Asia) for the confirmation of FMD. The systematic necropsy was performed within 12 h after the death of the animals. Representative tissue samples from the tongue, heart, trachea, lungs, liver, kidneys, thymus, palatine tonsils, spleen, mesenteric lymph nodes (MLN), stomach, pancreas, thyroid, adrenal glands, and different segments of the small and large intestines were collected in 10% NBF, and fixed for 4 to 5 days. Afterwards, the fixed tissues were processed for histopathological examination as per the standard procedure. Duplicate sections of each organ were used for immunohistochemical labeling of the FMDV antigen and a molecular confirmation of the presence of the virus. Similar types of tissues were collected from 4 apparently healthy slaughtered calves (cattle-2 and buffalo-2) for histopathological and immunohistochemical comparison. The tongue and heart tissues of these 37 dead calves were also screened for disease differentials such as Bovine Herpes virus (BHV4 &5), Bovine viral Diarrhoea virus (BVDV1&2), Malignant catarrhal fever (MCF), Bluetongue virus (BTV), Rabies, Pseudorabies, Listeria, Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida. All these pathogens were found negative.

2.2. Serum biochemical analyses

Ten milliliters (ml) of blood were taken from the naturally infected calves (n = 15) as well as FMDV recovered calves (1 month post infection) (n = 15) by jugular vein puncture in the BD Vacutainer® serum tubes for the separation of serum. Ten ml of the blood was collected from the apparently healthy calves (n = 6) for the comparison. Serum samples were harvested by centrifugation at 3000 rpm for 10 min and preserved at −20 °C until analysis.

The serum levels of blood glucose, triiodothyronine (T3), and thyroxine (T4) of FMDV infected and recovered calves were estimated with commercial kits (Coral diagnostics, PerkinElmer, USA) using bench-top semi-automatic biochemistry analyzer (Erba Chem 7, Germany). The serum level of thyroid Stimulating Hormone (TSH) was measured using commercially available ELISA kit (Sigma-Aldrich, USA). The level of cardiac troponin I (cTn-I) in the serum of FMDV infected, recovered, and control calves was estimated using commercially available ELISA kit (BlueGene Biotech, Shanghai).

2.3. Histopathology

Tissue sections were embedded in paraffin, sectioned at 4–5 µm, and stained with hematoxylin and eosin as per standard procedures (Luna 1972).

2.4. Immunohistochemical studies

To detect the FMDV antigen in the affected tissues, the duplicate paraffin sections from the above-mentioned organs and from 10 RT-PCR positive FMDV affected calves (cattle calves-6; buffalo calves-4) and four healthy calves were used for IHC technique. An indirect immunoperoxidase technique (IPT) was used to demonstrate the presence of the FMDV antigen. The paraffin sections were mounted on poly-L-lysine-coated slides (Sigma, St Louis, MO, USA). After deparaffinization and rehydration, endogenous peroxidase activity was quenched with 3% H₂O₂ in 80% methanol for 30 min. The sections were washed thrice in phosphate-buffered saline-Tween (PBST, 0.05 M, pH 7.6) for 5 min each. The slides were kept in citrate buffer (0.1 M, pH 6.0) and heated in a microwave oven for 15 min to unmask the antigen. The sections were washed in PBST followed by incubation in a humidified chamber with 1:10 normal goat serum in PBS with 1% bovine serum albumin (BSA) at 37 °C for 1 h to block non-specific sites in tissues. The blocking serum was drained out and replaced with FMDV Polyprotein (3D polymerase) primary antibody (bs-4524R, Bioss antibodies: 1:100 optimal working dilution), and incubated overnight at 4 °C. Sections were thoroughly washed in PBST thrice to remove unbound antibodies. Afterward, the sections were incubated with goat anti-rabbit IgG antibody horseradish peroxidase (HRP) conjugated secondary antibody (12-348, 1:200 dilution, Sigma-Aldrich, USA) at 37 °C for 1 h. Three washes were performed in PBST, and sections were immersed in a freshly prepared solution of 3-3′-diaminobenzidine tetrahydrochloride (DAB) and H₂O₂/urea tablets (Sigma) in distilled water. Sections were incubated at 37 °C for 45 sec or until the sections turned light brown in color. The slides were washed in running tap water to stop the reaction and counterstained with Mayer’s hematoxylin for 1 min. The sections were dehydrated, cleared, and mounted. For each batch of staining, test sections were also incubated with 10% solution of normal goat serum in PBS as a negative control. Immunoperoxidase scoring was performed by quantifying the positively stained cells observed in 8–10 different areas visualized under a 40× microscope objective. The scores were assigned as: 0 (none), absent; + (mild), few immunopositive cells; ++ (moderate), focally prominent immunopositivity; +++ (intense), strong immunopositivity in more than 50% of the cells. The negative control sections included the duplicate sections with the primary antibody substituted with a species-and isotype matched antibody, derived from the normal sera of rabbit (rabbit IgG isotype control, Novus Biologicals, USA). Additionally, the tissue sections from apparently healthy slaughtered buffalo calves were used for the negative control sections. For positive control, 10% NBF fixed tissues of tongue and heart tissues of cattle calves died earlier from being of naturally infected with FMDV (confirmed by multiplex-PCR and sandwich ELISA in tongue and heart tissues homogenates).

2.5. RNA extraction and cDNA synthesis

Viral RNA was extracted from the frozen tissues of the tongue and heart of 37 dead calves using the RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted RNA was further purified and treated with DNase I using an RNeasy kit (Qiagen, Hilden, Germany), and it was then quantified using a NanoDrop™ 1000 spectrophotometer to determine the OD₂₆₀/OD₂₈₀ ratio, which was 1.8–2.0 for all samples. The total RNA was stored at −80 °C until use. The cDNA synthesis was performed using a Revertaid First-Strand cDNA synthesis kit (ThermoFischer Scientific, USA). RT-PCR was used to detect the 5′ UTR of a FMDV gene using previously published universal primers specific for all serotypes (Reid et al. 2000). The amplified RT-PCR products were resolved on a 2% agarose gel by electrophoresis and were visualized by ethidium bromide staining. Multiplex PCR (MP-PCR) reactions were carried out for the detection of serotypes using published primers in the previous report (Giridharan et al. 2005). The MP-PCR include a universal FMDV-specific NK-61 primer (5′-GACATGTCCTCCTGCATCTG-3′, negative-sense) and three serotype-specific forward primers. These forward primers included DHP13, DHP15, and DHP9, which target the O, A, and Asia1 serotypes, respectively. The PCR reaction mix contained l µl of RT product, l µl of 10× PCR buffer, 0.6 µl of 25 mM MgCl₂, 0.4 µl of 10 mM dNTP mix, 4 pmols each of the virus-specific and serotype-specific primers, 0.25 U of HotstarTaq DNA polymerase, and 3.6 µl of RNase-free water. The thermal profile was one cycle of denaturation (95 °C for 15 min), followed by 30 cycles of denaturation (95 °C for 30 s), annealing (60 °C for 30 s), and extension (72 °C for 30 s). The reaction was completed with one cycle of a final extension (72 °C for 7 min). The amplicons were visualized by running the samples on a 2% agarose gel stained with ethidium bromide. Serotypes were differentiated based on amplicon size. Specifically, 249, 376, and 537 bp corresponded to the O, A, and Asia1 serotypes, respectively.

2.6. Statistical analyses

The levels of glucose, T3, T4, TSH and cTn-I in the serum of FMDV infected, recovered, and the control calves were measured by one-way ANOVA was done using Tukey’s post hoc test. The results were presented as mean (x) ± standard deviation (SD). The highly significant level was determined at p < 0.0001. All statistical analysis was performed using the GraphPad prism 8.0.1 statistical software.

3. Results

3.1. Clinical signs

All cases described in this study occurred in a well-organized dairy farm in Bareilly, Uttar Pradesh, India. The affected calves (59 cases) showed clinical signs of high fever (> 103.5 °F), anorexia, ropy salivation, stiff gait, flaccid paralysis (10 cases), tachycardia (> 105 beats/min), and tachypnoea (>58 breaths/min). Some animals displayed hyperemic buccal mucosa and mild catarrhal stomatitis (16 cases), in addition to vesicles on the oral mucosa of the lips (7 cases), cheeks (3 cases), gums (5 cases), hard palate (4 cases), dental pad (2 cases), and the rostral portion of the dorsum of the tongue (12 cases), muzzle (3 cases) and nares. The vesicles were fine-to-large bullae size (up to 6 cm in diameter). At times, they ruptured and formed red and raw ulcers. The feet in the majority of cases (18 cases) displayed swelling with blanched interdigital spaces and erosive vesicles. Out of 59 affected cases, only 37 calves died. Out of 37 dead calves, 8 calves (bovine-6, buffalo-2) died suddenly without showing any clinical signs of salivation, or lesions of blisters in the mouth and feet. A total of 19 (bovine-14, buffalo-5) calves died after 4 to 5 days of development of vesicular/blister lesions in the mouth or feet accompanied with a high rise of body temperature, while 6 calves (cattle-4, buffalo-2) died within 2 to 3 days after pyrexia and development of vesicular lesions. The episode of FMD was confirmed by viral detection methods that included RT-LAMP, sandwich ELISA, and multiplex RT-PCR (mRT-PCR) at the ICAR-Directorate of Foot-and-Mouth disease (DFMD, Mukteswar, India, and FAO Reference Laboratory for FMD for South Asia).

3.2. Serum biochemical analyses

The levels of blood glucose, cTn-I, T3, T4, and TSH in control, FMDV infected and recovered calves were presented in Figure 1(a–e). The level of glucose level (mg/dl) in the FMDV infected and recovered calves were significantly higher (p < 0.0001) as compared to the control calves (Figure 1(a)). The levels of cardiac cTn-I (ng/ml) (Figure 1(b)), T3 (nmol/L) (Figure 1(c)), and T4 (nmol/L) (Figure 1(d)) were significantly higher (p < 0.0001) in FMDV infected and recovered calves as compared to control calves. The TSH levels were significantly lower (p < 0.0001) in both FMDV infected and recovered calves as compared to the control calves (Figure 1(e)).

Figure 1. The FMDV infected and recovered calves showing higher serum levels (p < 0.0001) of glucose (a), cTn-I (b), T3 (c), T4 (d), and reduced levels of TSH (e) as compared to control calves. The datas were analyzed by one-way ANOVA with bonferroni post hoc test using GraphPad prism 8.0.1 statistical software.

3.3. Macroscopic findings

The affected calves displayed vesicle and ulcer formations in the buccal mucosa, the tongue, and the interdigital space of the hooves. The oral mucosa was congested and edematous in almost all cases. Erosive ulcers were found on the inner lower lip, dental pad, and the dorsal and rostral surface of the tongue in about 60% of cases. During necropsy, the abdominal and thoracic cavities of these animals contained 25–30 ml of clear-straw ascitic fluid (15 cases; cattle-12, buffalo-3). The hydropericardium was associated with pericardial hemorrhages, which were accompanied by petechiae or ecchymoses on the epicardial surface. The heart was flabby and dilated. The left ventricles had circular (1–4 mm in diameter) and linear (1–5 mm in length) grey-to-whitish necrotic streaks (arrow) interspersed with a normal unaffected myocardium. This produces a ‘tigroid heart appearance’ in the majority of calves (Figure 2(a,b)). Necrotic changes were pronounced in the interventricular septum of the affected calves (26 cases; cattle-21, buffalo-5), but these changes were also observed in the right ventricular free wall (5 cases; cattle-3, buffalo-3) and papillary muscles (4 cases; cattle-3, buffalo-1).

Figure 2. Gross and histopathological changes in various tissues of calves naturally infected with FMDV. a) Heart, cattle calf, 2.5 M. The ventricular myocardium showing necrotic grey to white streaks of variable size and shape giving ‘tigroid heart’ appearance. b) cross-sectional view of heart showing necrotizing myocarditis. c) Thymus, cattle calf, 4 M: swollen thymus with a few pin-head size blood spots on its surface. d) Lymph node, buffalo calf, 4 M. Cut surface of the lymph node showing severe congestion. e) Tongue, cattle calf, 3 M. Ballooning degeneration of stratum spinosum accompanied with severe inflammatory reaction. Hematoxylin and eosin (HE), X100. f) Heart, buffalo calf, 4 M. Heart showing acute interstitial necrotizing myocarditis characterized by marked infiltration of mononuclear cells (arrow). HE, X200. g) Lymph node, calf, 4 M. Depleted lymphoid follicles (arrow) in the cortex. HE, X100. h) Thymus, cattle calf, 4 M. Severe engorgement of blood vessel with the infiltration of inflammatory cells in the capsule of thymus. HE, X100. h) Small intestine, cattle calf 5 M. Enteritis showing marked infiltration of inflammatory cells in the lamina propria. HE, X100. j) Spleen, cattle calf, 4 M. Depleted lymphoid follicles in the white pulp region. HE, X100. k) Pancreas, buffalo calf, 4 M. Multifocal interstitial pancreatitis characterized by infiltration of inflammatory cells (arrow) surrounding the pancreatic acinar cells. HE, X100. l) Thyroid, cattle calf, 4 M. Presence of few inflammatory cells with scant colloid in the thyroid follicle. HE, X100.

Marked pulmonary congestion, edema, and focal hemorrhages were observed in the lungs in the majority of cases (32 cases; cattle-26, buffalo-6). The air passages were moderately congested and had frothy exudates. The thymus was swollen and flabby, displaying the presence of petechiae on the surface (4 cattle calves) (Figure 2(c)). The spleen was mildly swollen, and pin-sized hemorrhagic spots were found on its surface (5 cases; cattle-4, buffalo-1). The liver was swollen, mottled, and firm in consistency with a distended gall bladder (14 cases; cattle-11, buffalo-3)). Mild focal subcapsular hemorrhages were found in the livers of two cattle calves. The fundic and pyloric regions of the abomasum were markedly congested and edematous, and two cattle calves displayed paint-brush-like hemorrhages. The mucosa of the small intestine was severely congested and edematous, and it had diffuse and patchy congestion/hemorrhage over its serosa in 21 calves (cattle-17, buffalo-4), whereas the congestion of large intestine mucosa was observed in 4 cattle calves. The lesions were more severe in the small intestine compared to the large intestine. The MLN were severely edematous, enlarged, and congested (Figure 2(d)) in 22 cases (cattle-15; buffalo-7). The palatine tonsils, thyroid, adrenal glands, and pancreas showed mild petechial hemorrhages. The gross lesions were more prominent in cattle calves as compared to bubaline calves.

3.4. Histopathological findings

The gross and microscopic findings in FMDV-infected calves are summarized in Table 1. The mucosal epithelium of the lips and tongue showed ballooning degeneration, intercellular edema, numerous micro-vesicles and a few bullae formation, and necrotizing inflammation in the stratum spinosum and stratum basal layers (Figure 2(e)). The sub-epithelial tissues were edematous and congested, and revealed infiltration by mononuclear cells. In 8 cases, neutrophilic exudates were found to cover the entire tongue epithelium. The most conspicuous pathological findings in the affected hearts of both the species (37 calves) showed marked edema, congestion, hemorrhages, interstitial infiltration of mononuclear cells, hyaline degeneration and necrosis of cardiomyocytes (Figure 2(f)). Myocardial degeneration and necrosis vary from patchy and multifocal to confluent in all cases. The degenerated myocardial fibers were swollen, displaying eosinophilic and amorphous cytoplasm. Elongated nuclei had clumped chromatin and pyknosis in the foci of myocarditis and in adjacent sites. Pericarditis was characterized by edema and infiltration of mononuclear cells surrounding the pericardium in 6 cattle calves. In 4 cases, endocarditis was characterized by the infiltration of mononuclear cells surrounding the Purkinje fibers. Interestingly, the cells lining the tonsillar crypts showed vacuolar degeneration/necrosis and infiltration by mononuclear cells. The tonsillar crypts also displayed the presence of cellular debris in their lumen. The cryptic cells and the lymphoid follicles of the palatine tonsils underwent lymphoid depletion in 19 cases. The cortical lymph nodes showed marked lymphoid depletion (Figure 2(g)) with severe congestion, and hemorrhages. The capsule covering the thymus was markedly congested with infiltration of mononuclear cells (Figure 2(h)). The thymus showed thymic hyperplasia in 8 cases and mild depletion of cortical lymphocytes in 5 cases. Hassall’s corpuscles showed the infiltration of inflammatory cells in the epithelium and congested vessels in the parenchyma in 6 cases (cattle-5, buffalo-1). The small intestine showed severe enteritis characterized by marked infiltration of mononuclear cells in the lamina propria (Figure 2(i)). Peyer’s patches of the small intestine showed lymphoid depletion (lymphocytolysis) in 7 cattle calves and 25 cases (cattle-22, buffalo-3), respectively. However, in 7 cases (cattle-6, buffalo-1), reactive germinal centers in the cortex and severe macrophage reactions of the medullary sinuses were observed. The spleen displayed severe red pulp congestion and hemorrhages in 8 cases (cattle-6, buffalo-2), and moderate to severe lymphoid depletion of white pulp in 19 cases (cattle-15, buffalo-4) (Figure 2(j)).

Table 1. Frequency of FMDV induced lesions in different organs of 37 young cattle and bubaline calves affected with FMDV under natural condition.

Sl No.	Type of Organs	Gross lesions Number (%)	Microscopic lesionsNumber (%)
1	Tongue	22 (59.45)	27 (72.97)
2	Heart	35 (94.59)	37 (100)
3	Palatine Tonsil	14 (37.83)	21 (56.75)
4	Thyroid	0	3 (8.10)
5	Adrenal	0	5 (13.51)
5	Thymus	6 (16.21)	11 (29.72)
6	Mesenteric lymph node	22 (59.45)	25 (67.56)
7	Spleen	5 (13.51)	19 (51.35)
8	Pancreas	0	12 (32.43)
9	Lungs	32 (86.48)	33 (89.18)
10	Liver	10 (27.02)	20 (54.05)
11	Abomasum	7 (18.91)	17 (45.94)
12	Small intestine	21 (56.75)	29 (78.37)
13	Large intestine	4 (10.81)	7 (18.91)
14	Kidneys	4 (10.81)	9 (24.32)

The pancreas displayed multifocal interstitial pancreatitis (Figure 2(k)), which consisted of perivascular and interstitial infiltration of mononuclear cells in 12 cases (cattle-10, buffalo-2). Foci of inflammatory cells, degeneration of pancreatic acinar cells, interlobular, intralobular, and pancreatic duct’s connective tissue edema, dilation of the pancreatic excretory ducts, and degeneration of the lining of the epithelium of the ductal system were observed (4 cattle calves). In 3 cattle calves, necrosis was noted in the endocrine hormone- secreting islets of Langerhans cells. The thyroid glands showed mild infiltration by mononuclear cells within the follicles (thyroiditis), in addition to the presence of scant colloids in 3 cattle calves (Figure 2(l)). The adrenal glands revealed adrenalitis in 5 cattle calves, which was characterized by mild-to-moderate infiltration of inflammatory cells (especially mononuclear cells in the cortices and the medullae). The lung specimen showed diffuse and severe pulmonary congestion and edema, which was accompanied by focal intra-alveolar hemorrhage in more than 80% of cases (33 calves; cattle-26, buffalo-7). Ten infected cattle calves displayed moderate-to-severe interstitial pneumonia with thickening of the interalveolar septa, an increase in the number of macrophages, and perivascular infiltration by lymphocytes and plasma cells. Significant damage to the bronchial mucosa, significant inflammatory cell infiltration, and damage to the tracheal glands were observed in the sub-epithelial connective tissue stroma. In twelve cases, the liver showed mild portal lymphohistiocytic inflammatory infiltrates, multifocal-to-extensive hepatocellular necrosis, and increased activity in the Kupffer cells, which was coupled with severe congestion and hemorrhage. The hepatocytes showed fatty changes in 3 cases (cattle-2, buffalo-1) and hydropic degeneration in 12 cases (cattle-8, buffalo-4). However, in 5 cases, centrilobular necrosis of hepatic cells was also present. The moderate- to- severe infiltration of mononuclear cells was noticed around the portal triad in 5 cases (cattle-3, buffalo-2). The abomasum mucosa showed edema, congestion, and hemorrhages with an increased proliferation of lymphoid aggregates in 17 calves (cattle-14, buffalo-3). The kidneys showed moderate- to- severe congestion of capillaries in the cortex and medulla, and degeneration of the tubular epithelial cells in 8 cases. In 29 cases (cattle-24, buffalo-5), the small intestines showed goblet cell hyperplasia, shortening of the villi with degenerated enterocytes, dilation and necrosis of the intestinal crypts, and moderate- to- severe infiltration of mononuclear cells within the lamina propria, besides the lymphoid depletion in Peyer’s patches. The large intestine showed degenerated enterocytes with moderate infiltration of inflammatory cells in the lamina propria in 7 cases (cattle-6, buffalo-1). The microscopic changes were severe and predominant in cattle calves as compared to bubaline calves.

3.5. Immunohistochemical findings

A positive IHC reaction was characterized by the presence of light or dark brown, fine-to-coarse granules inside the cells and tissues of ten calves (both cattle and buffalo calves). This was complementary to the histopathological findings. The immunohistochemistry results obtained in both cattle and bubaline calves are presented in Table 2. The distribution and intensity of FMDV antigens in various organs of cattle calves were more as compared to buffaloes. The tongue showed an accumulation of the FMDV antigen in degenerated keratinocytes and in the areas of epidermal necrosis. An abundance of viral antigens was found in the cluster of individual cells located in the stratum basale and stratum spinosum layers in all the affected calves (Figure 3(a)). At the junction between the affected and normal epithelium, immunoreactivity was observed in individual or small clusters of basal cells lining the dermal papillae. Viral antigens shown by granular cytoplasmic intense brown- colored signals were present in the stratum basal layer in the absence of gross and histopathological alterations (Case #4–6, 8, 9). Additionally, macrophages in the dermis and a few cardiomyocytes were immunopositive in three cases (Case #1, 4, 7). Necrosed and degenerated muscle fibers in the heart showed dark brown-coloured cytoplasmic granular staining for the FMDV antigen in affected cattle and buffalo calves (Figure 3(b)). Positive moderate immunostaining was also observed in the subendocardial Purkinje fibers of the endocardium in 4 cases (Case #2, 5, 7, 8). The inflammatory exudates surrounding the pericardium also showed moderate immunoreactivity in three cases (Case #4, 7, 9). In the palatine tonsils, single cells or clusters of FMDV-positive cells were detected in all of the necrotic stratified squamous epithelial cells, in addition to the macrophages infiltrating the tonsillar cryptic epithelium. The lumen of the tonsillar crypts containing necrotic cellular debris also showed strong granular cytoplasmic immunoreactivity for viral antigens (Figure 3(c)). In the thymus, mild cytoplasmic light brown-coloured immunoreactivity was observed in Hassall’s corpuscles (Figure 3(d)) in 5 cattle calves, while absent in bubaline calves. In the MLN, positive staining was localized to the cytoplasm of macrophages in the medullary sinuses, while mild immunoreactivity was noticed in bubaline calves. The spleen showed mild-to-moderate immunopositivity within the germinal centers of the lymphoid follicles in 4 cattle calves (Case #2, 4, 5, 7) and one buffalo calf (Case #8).

Figure 3. The immunolocalization of FMDV antigen in various tissues of FMDV affected calves showing a) tongue showing distinct intense cytoplasmic immunostaining in the stratum basal and spinosum layer of cattle calf. The density of reaction is much higher in the stratum basal layer, IHC, X100. b) Distribution of FMDV antigen diffusely in multifocal manner in the necrosed cardiac muscle fibers of buffalo calf, IHC, X100. c) The macrophages infiltrating the tonsillar crypt epithelium showing the presence of FMDV antigen, IHC, X100. Note the presence of viral antigen in the necrotic cellular debris. d) Thymus showing cytoplasmic immunoreactivity for FMDV antigen in hassall’s corpuscles of cattle calf, IHC, X200. e) Pancreas showing high amount of FMDV antigen in the islets of Langerhans of buffalo calf. Note the moderate cytoplasmic immunoreactivity in the few pancreatic acinar cells, IHC, X100. f) Thyroid showing intense immunostaining for FMDV antigen in the follicular cells of cattle calf, IHC, X100. g) Adrenal gland showing diffuse granular strong cytoplasmic staining in zona reticularis layer of the adrenal cortex of cattle calf, X200. h) The small intestinal epithelial cells as well as the intraepithelial inflammatory cells showed the presence of FMDV antigen of buffalo calf, IHC, X100. i) The tubular epithelial cells of the kidney in cattle calf showing cytoplasmic immunopositivity for FMDV antigen, IHC, X100.

Table 2. Severity and localization of the immunohistochemical findings in various tissues of cattle and buffalo calves naturally affected with FMDV.

Immunohistochemical Findings
Case No.	Species	Age	Breed	Sex	Tongue	Heart	Tonsil	Thyroid	Thymus	Lungs	Liver	Abomasum	Pancreas	Spleen	Mesenteric lymph node	Small intestine	Large intestine	Adrenal	Kidneys
1	Virindavani(cattle calf)	4m	Cross bred	Female	+++	+++	++	+++	+	+	–	+	++	–	++	++	+	+++	+
2	Virindavani (cattle)	4m	Cross bred	Female	++	+++	++	++	+	–	+	–	++	+	++	+	–	++	+
3	Virindavani (cattle calf)	3.5m	Cross bred	Female	+++	+++	+++	+++	–	+	–	–	+++	–	+	++	++	+++	++
4	Virindavani (cattle calf)	2m	Cross bred	Male	+++	++	+++	+++	++	–	+	–	++	+	++	++	+	++	+
5	Virindavani (cattle calf)	4.5m	Cross bred	Female	++	+++	++	++	–	++	–	–	+++	++	++	++	–	+++	++
6	Virindavani (cattle calf)	4m	Cross bred	Female	+++	+++	+	–	–	+	+	–	++	–	++	+	+	++	–
7	Virindavani (cattle calf)	3m	Cross bred	Male	++	++	++	+++	+	++	–	+	++	++	+++	++	+	+++	++
8	Murrah (buffalo calf)	1m	Pure	Male	+++	+++	+	–	–	+	–	–	++	+	+	+	–	–	+
9	Murrah (buffalo calf)	1m	Pure	Female	++	++	++	+	–	–	–	–	++	–	+	+	–	++	–
10	Tharparkar (cattle calf)	3m	Pure	Male	++	+++	++	++	+	–	–	–	+	–	++	++	++	++	–

The distribution of viral antigens within the endocrine glands was noteworthy. In the pancreas, the FMDV antigen was detected in the plasma membrane and cytoplasm of epithelial cells of the acini, interlobular ducts, and islets of Langerhans. The presence of viral antigen was more pronounced in almost all of the islets of Langerhans of all 10 affected calves (Figure 3(e)). Numerous small follicular cells of the thyroid showed distinct cytoplasm immunolabeling for the viral antigen in 7 cattle calves (Case #1–5, 7, 10) and 1 case of buffalo calf (Case # 9) (Figure 3(f)). The zona reticularis of the adrenal glands showed intense dark brown coloured cytoplasmic immunoreactivity in all cattle calves, while 1 bubaline calf showed moderate immunoreactivity (Case #9) (Figure 3(g)). The chromaffin cells of the medulla displayed moderate immunostaining in 4 cattle calves. However, 4 cases revealed intense cytoplasmic immunopositivity for the viral antigen in both the zona fasciculata and zona glomerulosa of the adrenal glands (Case #1, 3, 5, 7). In the lungs, alveolar and bronchial/bronchiolar luminal fluid showed immunopositivity for the FMDV antigen in 5 cattle calves and I buffalo calf, and faint immunolabeling was noticed in a few alveolar macrophages of cattle calves. In the trachea, moderate immunoreactivity was noticed in the tracheal glands in 4 cases (Case #2, 4, 8, 10). In 10 cattle calves, moderate cytoplasmic immunolabeling was detected in the cytoplasm of the cryptal epithelium, in addition to the mononuclear cells in the lamina propria of the small intestine (Figure 3(h)), while mild immunoreactivity was observed in 2 bubaline calves. The large intestine showed mild -to moderate immunoreactivity in infiltrating mononuclear cells of the lamina propria in 5 affected calves, while absent in bubaline calves. The cells lining the renal pelvis and proximal convoluted tubules showed mild-to-moderate accumulation of the viral antigen in 6 cattle calves (Case #1–5, 7) (Figure 3(i)) and 1 buffalo calf (Case # 8). Except for weak staining in the bile duct epithelium, the hepatocytes displayed no immunostaining in 3 cattle calves (Case #2, 4, 6). Similarly, the crypts of the abomasum showed mild cytoplasmic staining in two cases (Case #1, 7). There was no immunostaining observed in the control tissue sections obtained from apparently healthy slaughtered buffalo tissues and isotypes matched antibody.

3.6. Molecular confirmation of FMDV

Positive RT-PCR results consisted of the amplification of a 328-bp genome fragment in the tongue and heart tissues of 37 calves. The amplified products from all tissues displayed 100% nucleotide sequence homology to each other. A maximum identity of 100% was obtained for the Bangladesh strain BANGA-SA-197, which belongs to serotype ‘A’ with genotype VII. A maximum identity of 99% was also obtained for the Saudi Arabian SAU/2015, which belongs to serotype ‘A’. Furthermore, FMDV serotype ‘A’ was confirmed using multiplex RT- PCR by amplifying a 376-bp genome fragment (Figure 4).

Figure 4. Molecular detection of FMDV type A by multiplex RT-PCR in tissue samples of calves. Lane M–100 bp + DNA ladder, N-negative control, Lane 1–7 amplicons positive for Type A (376 bp), L1-Tongue, cattle calf, case 1, L2-Heart, cattle calf, case 4, L3- Tongue, buffalo calf, case 8, L4- Heart, case 9, L5- Tongue, buffalo calf, case 9, L6- Heart, buffalo calf, case 9, L7-Heart, cattle calf, case 7.

4. Discussion

The current study is the first account of describing the tropism of FMDV in various organs of bovine calves under natural conditions. This is of particular importance because of the novel pathological and immunohistochemical localization of this virus in different organs of calves aside from its normal target locations. The clinical signs and gross lesions on the tongue and heart, and the significant immunoreactivity for viral antigen are consistent with earlier research (Gulbahar et al. 2007; Ryan et al. 2008; Stenfeldt et al. 2014; Aktas et al. 2015; Ranjan et al. 2016). Our recent findings are strikingly similar to earlier descriptions of the vesicular features and myocardial lesions brought on by EV-71 and Coxsackieviruses of the Enterovirus genus (which are other types of Picornaviruses) (Muehlenbachs et al. 2015; Koh et al. 2016). In instance, 25–40% cases of acute myocarditis and dilated cardiomyopathy in infants and young adolescents have been linked to the Coxsackievirus group B (CVB3) (Akuzawa et al. 2014).

The high serum levels of glucose in both affected and recovered calves than the control calves in the present study are in congruent with the earlier observations which suggest the damage to the islets of Langerhans due to replication of FMDV causing hyperglycemia due to destruction of insulin producing cells (beta cells of pancreas) (Ghanem and Abdel-Hamid 2010; Mousa and Galal 2013; Salim et al. 2019). The higher levels of T3, T4 levels and reduced levels of TSH suggest the hyperthyroidism due to the damage of thyroid gland leading to the release of the stored hormones due to damaged follicular cells of thyroid rather than altering the negative feedback regulation. The similar finding was recorded in an earlier report (Saravanan et al. 2020). The high values of cTn-I in the serum of affected and diseased calves suggest the myocardial injury due to FMDV. The serum level of cardiac troponin concentration was regarded as earlier marker of myocardial injury after virus infection than microscopic lesions of myocarditis (Lim et al. 2005). The similar high rise of serum cTn-I level was reported in previous reports (Tunca et al. 2008; Aktas et al. 2015; Aly et al. 2020; Saravanan et al. 2020; Mahadappa et al. 2021).

The lymphocytolysis and immunoreactivity for viral antigens in lymphoid organs (palatine tonsils, spleen, thymus, MLN, and Peyer’s patches) suggest that the infection may be immunosuppressive as a result of the secondary phase of viral replication in the lymph nodes. Moreover, the presence of infected immune cells may represent a non-cardiac reservoir, which could play an important role in the dissemination of the virus and in the persistence of the infection (Klingel et al. 1996). Previous descriptions have revealed significant lymphopenia and lymphoid depletion during FMDV infections (Golde et al. 2011; Eschbaumer et al. 2016). However, this is in contrast to one report describing the immunocompetence of cattle during the acute period of infection (Windsor et al. 2011). The presence of distinct cytoplasmic immunolabeling for viral antigens in the crypt epithelium and macrophages of the palatine tonsils supported the presence of virus in the tonsillar macrophages. From there, they probably disperse via the lymphatic system into the circulation. This is consistent with previous studies, which have shown the localization of FMDV in the tonsils of adult cows using immunomicroscopy (Arzt et al. 2010; Stenfeldt et al. 2015; Ranjan et al. 2016) or in situ hybridization (Brown et al. 1996; Durand et al. 2008) during the acute and persistent phase of infection. Previous report supports the view that the Enteroviruses infect the immune cells. This plays a pivotal role in acute or persistent cases of myocarditis (Klingel et al. 1996).

This study revealed the localization of the FMDV in the endocrine glands, in addition to the pancreas, thyroid gland, and adrenal glands. The results of FMDV-induced pancreatic damage have been clearly documented in earlier reports (Brown et al. 1991; Portiansky and Gonzalez 1995; Sanz-Ramos et al. 2008). However, no studies have demonstrated the presence of viral antigens within the cell types of the pancreas. In this study, FMDV antigens were consistently demonstrated in both pancreatic acinar cells and the islets of Langerhans. This is a finding not previously reported. The necrosis and the presence of abundant viral antigens in the islet suggest that FMDV might play pivotal role in the development of Type 1 diabetes. This is the result of the direct action upon beta cells, causing defective insulin production (Clark 2003). Various reports have described hyperglycemic condition in cattle infected with FMDV (Mohapatra et al. 2005; Ghanem and Abdel-Hamid 2010), which might be the result of the destruction of islet cells, which is appeared to be the case in this study. Moreover, the high levels of blood glucose in both FMDV infected and recovered calves in the present investigation support the same. The members of the Picornavirus family are similar to the Enteroviruses and, especially, the CVB. They have been implicated in 20–34% of all human pancreatitis cases (Mena et al. 2000; Chrysos et al. 2004; Zhang et al. 2016). The localization of enteroviral protein has been shown in several studies by employing immunohistochemistry (Skog et al. 2014; Muehlenbachs et al. 2015) and in situ hybridization techniques (Ylipaasto et al. 2004).

The increase in viral antigens within the developing follicular cells of the thyroid gland indicates that this is the most favorable site for viral replication. The presence of FMDV antigens in the thyroid without significant gross lesions could be ascribed to the fact that some RNA viruses allow the cell to survive to shed new virions over a long period without causing any appreciable changes (Psalla et al. 2006). Thus, even after recovery, the animals suffer from thyroid insufficiency, which might be caused by the prolonged replication period in the follicular cells of the thyroid. In our case, higher values of T3, T4, and reduced levels of TSH in case of recovered animals corroborate with the findings of earlier report (Saravanan et al. 2020). Additionally, it is suggested that natural reservoir hosts always maintain a non-pathogenic equilibrium with their virus to support active viral replication without the development of disease (Broussard et al. 2001). The presence of viral antigens in the thyroid gland leads us to hypothesize that the release of thyroid hormone into the bloodstream following membrane damage might result in the observed weight loss, tachycardia, and fatigue. Recent findings have described the presence of Enteroviruses in thyroid tissues of human patients (Hammerstad et al. 2013).

One of the important results of this study is the discovery of the localization of FMDV antigens in the zona reticularis and chromaffin cells in the medulla of the adrenal glands. This is the first report of this nature. Because the zona reticularis is involved in sex hormone production, the replication of FMDV suggests that the virus may reduce fertility. Moreover, the presence of viral antigens in the medulla may indicate the release of stress hormones (epinephrine and norepinephrine), which can lead to hypovolemic shock. This can manifest as a coma or even death in the advanced stage of disease. An earlier report has described infertility as a consequence of FMD infections in cattle (Honnappa 2014), which might be the result of the persistence of viral antigens in the adrenal gland as we observed in the present investigation.

In livestock, numerous studies have been carried out to understand the role of the lung in the pathogenesis of FMD. Some researchers argue that the lungs are the site of viral replication during the early phase of the infection (Brown et al. 1996; Arzt et al. 2010), whereas others have suggested that the lungs are not an organ of interest during the early replication and dissemination of the virus (Murphy et al. 1999; Stenfeldt et al. 2015). The low presence of antigens in the lungs indicated that either it might not be a primary site for the initial viral growth or FMDV might have been cleared from the lungs by alveolar macrophages. The presence of intracytoplasmic FMDV antigens in alveolar macrophages is likely the result of phagocytosis of FMDV-infected cells and might represent a mechanism of viral persistence as a result of protection from neutralizing antibodies. However, the presence of FMDV in the epithelial cells lining the bronchi, bronchioles, trachea, and tracheal glands is important because it suggests a mechanism of viral transmission to other susceptible animals. Therefore, infective shedding, excretion, and secretion would be increased in animals infected with FMDV.

The presence of immunopositive staining in the renal tubular epithelial cells, cortical vessels, and transitional epithelia of the renal pelvis suggested that the virus might have reached the kidneys via the bloodstream, passed through the glomerular filtrate, and underwent excretion in the urine. This might play a key role in the spread of the virus. This observation is congruent with an earlier report describing the excretion of the virus in the urine of cattle, which aids the spread of this disease (Alexandersen et al. 2003).

The presence of FMDV antigens in the epithelial cells of the villi, crypts, and mononuclear cells of lamina propria of small and large intestines confirmed the association of FMDV with enteritis.

Although the liver displayed a variety of pathological alterations (ranging from changes in fat content to centrilobular necrosis) and the stomach showed lymphoid hyperplasia, we failed to detect the presence of viral antigens in the liver or the stomach, except for weak staining in three and two cases, respectively. It is thought that because the liver and stomach are not the main target organs, the presence of viral antigen is therefore low. However, the evidence of hepatic injury is comparable to the findings of earlier studies that reported liver damage in FMDV-infected cattle (Ghanem and Abdel-Hamid 2010; Anil and Gurudutt 2011).

Like FMDV, enterovirus antigens were also found in the tongue, heart, pancreas, adrenal glands, renal tubular cells, hepatocytes, and lungs in various studies, suggesting broad tissue tropism (Yu et al. 2014; Muehlenbachs et al. 2015; Zhang et al. 2016).

In the present study, FMDV serotype A was responsible for causing this outbreak. The emergence of multiple genetically and antigenically divergent lineages and poor intergenotypic antigenic coverage within serotype A makes it difficult to control via routine vaccination (Rudreshappa et al. 2012). Thus, in the present study, the outbreak occurred in organized dairy farm despite regular vaccination programs. The currently used trivalent inactivated vaccine (serotypes O, A, and Asia1) in India comprised of 95% from serotype O and the remaining 5% from both serotypes (A and Asia 1). Moreover, it is reported that 75.6% field FMDV of serotype A showed antigenic drift from the existing serotype A vaccine strain (Type A IND 40/2000), thereby recommended the use of IND 27/2011 type A in vaccine formulation in recent reports (Mohapatra et al. 2018, Sreenivasa et al. 2021). As the FMDV outbreak is due to serotype A in the present outbreak, thus the currently used vaccine couldn’t give protection against the outbreak giving rise to high mortality despite regular vaccination.

5. Conclusion

In conclusion, the present findings revealed the novel pathological lesions and immunolocalization of FMDV antigens in the previously unreported tissues such as adrenal glands, thyroid, pancreas, spleen, lymph nodes, thymus, intestines, and kidneys for the first time. This is in addition to FMDV localization in the tongue and heart (earlier reported), suggesting its pathogenic potential and replicative fitness in various organs in calves. The pathological findings and the viral antigen distributions in the tongue, heart, pancreas, and adrenal glands of calves infected with FMDV are similar to those of enterovirus infections. This suggests similar mechanisms of infection are at work between these viruses and their hosts. Our results suggest that FMDV serotype A is a pantropic virus causing multi-systemic disease in calves. The clinical signs, gross and histopathological and FMDV antigen distributions were more pronounced in cattle as compared to buffaloes. Differences in the patterns of viral antigen distributions might be influenced by viral strains, date of sampling, and various host factors. Future experimental studies in both cattle and buffalo calves are needed to better understand the physiopathogenetic and immunopathogenic aspects of the disease in 2 different species. Using the indirect immunoperoxidase method, FMDV antigen detection can be achieved in formalin-fixed material. Therefore, this immunohistochemical method could be used to diagnose naturally occurring cases, especially in the absence of fresh tissue.

Acknowledgements

The authors are grateful to the Joint Director (Research) and Director of the Institute for providing the necessary facilities to carry out this work. The first author wishes to acknowledge the financial assistance provided by IVRI, Deemed University to carry out this work.

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Funding

The author(s) reported there is no funding associated with the work featured in this article.