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Patho-epidemiological and Immunohistochemical Study of Squamous Cell Carcinoma in Cattle and Buffaloes

R
Riddhi V. Trambadiya1
0009-0009-5430-5479
J
Jignesh M. Patel1,*
S
Samir H. Raval2
R
Rohit S. Parmar3
J
Jignesh B. Patel4
1Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar- 385 506, Gujarat, India.
2Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand-388 001, Gujarat, India.
3Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari-396 450, Gujarat, India.
4Department of Veterinary Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.

ABSTRACT

Background: Study was conducted to investigate the etiopathogenesis of squamous cell carcinoma (SCC) in cattle and buffalo through pathoepidemiological, histopathological, immunohistochemical and molecular analysis.

Methods: A total of 64 tissue samples of SCC were collected between December 2024 and December 2025.  Immuno histo chemistry (IHC) was performed to detect immunoexpression of pan cyto keratin (PCK), proliferating cell nuclear antigen (PCNA), zonula occludens-1 (ZO-1), E-cadherin, Vimentin, FOXA1, p120, GATA3, γ-H2AX and p63. Polymerase chain reaction (PCR) was performed to detect papillomavirus.

Result: Out of 64 cases, ocular tumors were the most prevalent, followed by horn cancer (34.38%) and vulvar tumors (9.37%). Cattle accounted for 87.5% cases, with crossbred Holstein-Friesian showing the highest occurrence. SCC was most frequently observed in females and in animals aged 7-10 years. Histopathologically, most tumors were well differentiated (WD) (70.31%). Immunohistochemically, SCCs exhibited strong expression of PCK, PCNA, p120, γ-H2AX and p63, while ZO-1 showed moderate expression. Upregulation of vimentin and downregulation of E-cadherin indicated epithelial-mesenchymal transition (EMT), whereas increased γ-H2AX expression suggested enhanced DNA double-strand damage in poorly differentiated tumors. PCR confirmed the presence of papillomavirus in four (6.25%) SCC cases.

 

INTRODUCTION

Cutaneous tumors are the most frequently diagnosed neoplasms in veterinary medicine (Goldschmidt and Goldschmidt, 2016). SCC is the most common malignant epithelial neoplasm in livestock and can arise at multiple anatomical sites viz. eye, horn, vulva, teat, udder, perineum and skin. In India, SCC accounts for approximately 80% of reported neoplasms in cattle and 3% in buffaloes (Somvanshi, 1991). Several risk factors have been implicated in the development of the tumor, such as chronic ultraviolet (UV) radiation, non-pigmented skin, lack of hair, chronic irritation, trauma, breed predisposition, age and sex (Velavan et al., 2025).
       
Immunohistochemistry was performed using a panel of biomarkers to evaluate different biological aspects of SCC, including PCK for epithelial origin, PCNA for cellular proliferation, ZO-1, E-cadherin and p120-catenin for cell adhesion and epithelial integrity, vimentin for EMT, γ-H2AX for DNA double-strand break, p63 for squamous differentiation and FOXA1 and GATA3 as transcription factors (Mestrinho et al., 2015; Karakurt et al., 2021). A limited number of studies have evaluated immunohistochemical biomarkers associated with proliferation, EMT and genomic instability in SCC in a single cohort.
       
Papillomaviruses are strongly associated with papillomas and SCCs across multiple species. However, the exact mechanisms involved in carcinogenesis remain poorly understood. This limits the understanding of tumor behavior, progression and diagnostic biomarker utility in farm animals.
       
The objectives of the present study were to investigate the pathoepidemiological and pathomorphological characteristics of SCC in cattle and buffaloes, characterize histopathological lesions, evaluate immunoexpression of selected biomarkers and determine the presence of papillomavirus using PCR.

MATERIALS AND METHODS

Study period and location
 
The research work was conducted at the Department of Veterinary Pathology, COVSAH, KU, Sardarkrushinagar, Gujarat between December, 2024 and December, 2025.
 
Sample collection
 
A total of 87 suspected samples were examined. Histopathological evaluation confirmed 64 cases as SCC, while 23 tissue growths were diagnosed as non-neoplastic and excluded from further analyses. Among 64 cases, 22 horn cancer (HC), 36 ocular SCC (OSCC) and 6 vulvar SCCs (VSCC) were noted (Supplementary Material 1). The tissue specimens were collected from 1) Veterinary Clinical Complex, COVSAH, KU, Sardarkrushinagar 2) Anchorwala Ahinsadham, Mundra and 3) Jalaram Gaushala, Bhabhar. Tissue was collected in 10% neutral buffered formalin (NBF) for histopathological analysis and in sterile containers for molecular analysis without any preservatives and stored at -20°C as soon as possible.

Supplementary Material 1: Details of samples collected (n=64).


 
Histopathology
 
All collected tissues were fixed in 10% NBF, subjected to standard processing. The entire tissue processing was accomplished with automatic tissue processor and subjected to hematoxylin and eosin (HandE) staining according to standard protocol of Luna (1968). SCCs were graded according to the Modified Anneroth’s grading system (Table 1).

Table 1: Modified anneroth’s grading system.


 
Immuno histo chemistry (IHC)
 
Histopathologically confirmed slides were subjected to immunohistochemical examination with various biomarkers using standard protocol of Raval et al., (2018). The information regarding primary antibody used in this study were mentioned in Table 2.  After TBS rinsing, sections were incubated with Envision rabbit/mouse reagent conjugated with peroxidase (Dako, Denmark) for 30 minutes. Chromogenic reaction was visualized using 3,3’-diaminobenzidine (DAB) and counterstained with Harris-hematoxylin, dehydrated, cleared and mounted.

Table 2: Details of Primary antibody used in IHC.


       
Immunohistochemical scoring was carried out by a semi-quantitative scoring system by assessing the percent of positive immunoreactive cells i.e. for PCK (Baghla et al., 2012; Kumar et al., 2025), for PCNA (Kumaraguruparan et al., 2006), for ZO-1 (Upadhaya et al., 2022), for E-cadherin (Pinho et al., 2007), for p120 (Rajeev et al., 2024), for γ-H2AX (Dörtbudak and Yener, 2025) and for p63 (Mestrinho et al., 2015). Strong nuclear immunoreactivity for FOXA1 and strong nuclear staining in ≥10% neoplastic cells were considered as positive to GATA3 biomarker (Machado et al., 2020).
 
Molecular detection of oncogenic virus
 
DNA extraction
 
The DNA isolation from tissues was accomplished using the commercially available DNeasy Blood and Tissue Kit manufactured by QIAGEN-GmbH, Germany according to the manufacturer’s instructions. All DNA was subjected to quality evaluation using highly conserved mammalian IGF-1 gene (Table 3).

Table 3: Specifications of primers.


 
Detection of Papillomavirus
 
Primer specific to papillomavirus were adopted from Forslund et al., (1999) (Table 3). Reaction mixture was prepared containing 10 μL of PCR mastermix, 0.8 μL of each primer, 2.4 μL template DNA and 6 μL PCR grade water. PCR was performed in thermal cycler (The Veriti® Applied Biosystems) according to protocol mentioned in Table 4. The detection of amplified products were performed in 1.5% Agarose gel at 85 voltages for 40 minutes and the DNA bands were visualized using Dual LED Blue/White Light Transilluminator and photographed.

Table 4: Protocol for PCR.

RESULTS AND DISCUSSION

Incidence
 
Current study reported that OSCC was the predominant tumour, followed by HC and VSCC (Table 5) and found similarity with an earlier study (Parmar et al., 2024). In cattle 56 cases were recorded while in buffaloes only 8 cases were found, that were similar to the findings revealed by Reddy et al., (2025). Among HC, 19(86.36%) were noticed in cattle whereas 3(13.64%) were diagnosed in buffaloes. Similarly OSCC and VSCC were reported higher in cattle as compared to buffaloes.

Table 5: Histopathological grading of SCC.


       
Highest incidence was noted in HF-crossbred cattle (44.64%) followed by Kankrej (25.00%), Non-descript (21.43%) and Gir (8.93%); found compatible with Yeruham et al., (1999) and Parmar et al., (2024). In buffaloes, equal distribution was reported among Mehsana(50%) and Banni breed(50%).
       
Highest incidence was observed in the 7-10 years age group(n=47), followed by <7 years(n=13) and the least in >10 years(n=4), which was comparable to Dave et al., (2025). However, Udharwar et al., (2008) reported higher occurrence in >10 years. There was equal incidence of HC among male and female. Higher occurrence of bovine OSCC was noted in females (86.11%) and were in agreement with Kalim et al., (2021) and Nithya et al., (2022).

Gross pathology of SCC
 
Gross pathomorphology of affected parts were recorded (Supplementary Material 2). In HC, majority cases (n=12) exhibited cauliflower shaped growth at base of affected horn; while others showed nodular growth with verrucous surface (n=10). Most growths had a firm consistency. Some large ulcerated growth showed bleeding from wound. Cut sections revealed yellowish-white, grayish-white, reddish-brown to brownish-black surface along with widespread hemorrhage and necrosis (Desai et al., 2024; Kumar et al., 2025).

Supplementary Material 2: Gross pathomorphological features of suspected cases of SCCs.


       
Majority OSCC (n=19) were found on nictitating membrane followed by lower eyelid, bulbar conjunctiva, corneo-scleral junction and upper eyelid, respectively. Majority had cauliflower like appearance followed by nodular growth with rough and verrucous surface. Few cases had papillomatous and pedunculated growth. Similar alterations were revealed by Fornazari et al., (2017), in contrast to Karakurt et al., (2023).
 
Histopathology of SCC
 
Histopathological grading was carried out and divided into WD, MD and PD tumors (Table 5). Scoring of individual tumorous mass was noted (Supplementary Material 3).

Supplementary Material 3: Histopathological scoring of individual SCC as per modified Anneroth’s grading system.


       
Histopathology of WD-HC revealed proliferating neoplastic cells forming concentric lamellae of keratins developing large epithelial pearls (Fig 1A). Prominent keratinization was observed in center of islands (Joshi et al., 2009). Cells were arranged in islands and showed moderate pleomorphism. One case showed a transition between pseudostratified columnar epithelium to SCC (Fig 1B) and also showed interconnected osseous trabeculae that supported by loose fibrovascular stroma (Fig 1C). Mitotic index was very low. MD neoplasms revealed neoplastic cells arranged in cords and trabeculae and showed pleomorphism and apoptosis.

Fig 1: Histopathology.


       
Well-differentiated OSCC revealed presence of round to polygonal shaped cells arranged in islands, cords and trabeculae, supported by fibrovascular stroma and typical keratin pearls (Fig 1D). Neoplastic cells had distinct cell borders and showed anisocytosis and anisokaryosis, karyomegaly, apoptosis and few mitotic figures (Fig 2A). Few cases revealed marked granulomatous inflammation characterized by infiltration of macrophages, multinucleated giant cells, plasma cells, lymphocytes and polymorphs cells (Fig 2B). WD and MD neoplasms showed multifocal neoplastic cell with prominent intercellular bridges (desmosomes) (Fig 2C). PD infiltrative neoplasm showed polygonal cells arranged in cords and trabeculae. Mononuclear cells infiltration and atypical mitotic figures were noted (Sharma et al., 2020; Karakurt et al., 2023).

Fig 2: Histopathology.


       
VSCC showed proliferating neoplastic cells arranged in small islands that formed concentric layers, developing multiple prominent characteristic epithelial pearls (Fig 2D). Neoplastic cells showed minimal anisocytosis and anisokaryosis (Khodakaram-Tafti et al., 2013).
 
Immuno histo chemistry of SCC
 
Immunohistochemical examination was performed using aforementioned various tumor markers. IHC revealed all cases of SCCs (100%) were positive for PCK, PCNA and p63; 42 (65.63%) for ZO-1; 35 (54.68%) for E-cadherin; 39 (60.94%) for p120; 52 (81.25%) for Vimentin; and 56 (87.50%) were for γ-H2AX. However, FOXA1 and GATA3 failed to react with any tumorous tissue. Immunohistochemical scoring of individual tumor mass for each marker is given in Supplementary Material 4.

Supplementary Material 4: Immunohistochemical scoring of individual SCC.


       
Current study reported that all cases showed cytoplasmic immunoreactivity to PCK. Out of 64, 4(6.25%) cases depicted immunoreactivity in >75% neoplastic cells and were considered strongly immunoreactive (Fig 3A). However, 25(39.06%) cases showed expression in 51-75% cells, 24(37.50%) in 26-50% cells and 11(17.19%) in <25% cells. WD-SCC showed strong cytoplasmic immunoreactivity with pancytokeratin in neoplastic cells and keratin pearls (Fig 3B). The present findings were similar to those of Kumar et al. (2023). The strong immunolabelling of PCK indicates tumor of epithelial origin.

Fig 3: Immunohistochemistry.



Among 64 cases, 7 had nuclear immunoreactivity to PCNA in >75% of neoplastic cells and were considered strongly immunoreactive. However, 26 cases showed expression in 51-75% cells, 28 showed expression in 26-50% cells and 3 showed expression in 5-25% of cells. These findings suggested that WD-SCC showed mild to moderate nuclear immunostaining (Fig 3C), while PD-SCC showed strong immnunostaining in proliferating neoplastic cells (Fig 3D) that suggests proliferative potential and biological aggressiveness of the tumor. These findings were in accordance with Martano et al., (2016). However, Karakurt et al., (2021) did not find any significant difference. 

       
Among 64 cases, only one case (1.56%) showed membranous immunostaining of ZO-1 in >75% neoplastic cells, 13(20.31%) in ≥50-<75% cells, 19(29.69%) in ≥25-<50 cells, 9(14.06%) in ≥10-<25 cells and 22(34.38%) were in <10% neoplastic cells. 54 cases showed weak immunostaining (Fig 4A), while 10 had moderate membranous staining (Fig 4B). All tumors exhibited low ZO-1 expression in current study (Qiu et al., 2016; Upadhaya et al., 2022), indicating disruption of tight junction integrity during malignant transformation.

Fig 4: Immunohistochemistry.


       
Out of 64 SCCs, 35(54.68%) cases showed positive immunoreactivity with E-cadherin (Fig 4C) and 29 were found non-immunoreactive. Among the 35 positively expressed tumors, 20 cases (20/35; 57.14%) showed immunolabelling in <25% neoplastic cells and 15(42.86%) cases showed in 26-50% neoplastic cells. Upadhaya et al., (2022) reported complete disruption of the adherens molecule in all cases. This down-regulation of E-cadherin indicates disruption of adherens junctions in epithelial tissues as cells progresses towards malignancy.
       
Among 64 SCCs, 52(81.25%) cases were found immunopositive for vimentin (Fig 4D), while 12 cases were found non-immunoreactive. This suggests that EMT is crucial for tumor invasion and metastasis. The present finding was found compatible with Yu et al., (2015).
       
FOXA1 did not show any nuclear immunoreactivity in neoplastic cells and was thus considered as non-immunoreactive (Fig 5A). This negative immunoreactivity could be due to the antibody clone failing to react with bovine tissue. Similarly, all cases were found negative for GATA3 immunoreactivity (Fig 5B). Liu et al., (2012) also reported similar findings in SCC cases.

Fig 5: Immunohistochemistry.


       
A total of 39(60.94%) cases showed p120 membranous expression in >50% neoplastic cells and were considered to show preserved expression of p120 (Fig 5C). Whereas, 25(39.06%) cases showed <50% of positive neoplastic cells and considered as reduced expression (Fig 5D). Li et al., (2020) also reported decreased expression in SCC, which may indicate loss of cell adhesion and enhanced invasive potential of neoplastic cells.
       
In this study, 56 (87.50%) were found immunoreactive for γ-H2AX whereas 8 (12.50%) were found negative. Out of 56, 31(55.36%) cases showed mild nuclear immunoreactivity (Fig 6A), 16(28.57%) showed moderate immunoreactivity and 9(16.07%) showed severe immunoreactivity (Fig 6B). While 8(12.50%) cases were found non-immunoreactive similar to Dörtbudak and Yener (2025). Chronic ultraviolet (UV) radiation exposure is considered an important etiological factor that can induce DNA damage, leading to increased γ-H2AX expression.

Fig 6: Immunohistochemistry.


       
Furthermore, all cases were found immunoreactive for p63 (Fig 6C) and (Fig 6D). A total of 15(23.44%) cases showed p63 expression in >70% neoplastic cells, 24(37.50%) cases in >50-<75% cells and 25(39.06%) cases in <25% neoplastic cells. In current study, p63 expression showed a significant correlation with the degree of tumor differentiation similar to Mestrinho et al., (2015).

Molecular detection of oncogenic virus
 
All extracted DNA tested positive on the quality-assessment using PCR for IGF-1 gene (Fig 7). Present investigation reported successful molecular detection of Papillomavirus in only four cases (6.25%) (Fig 8). Earlier studies were unable to detect BPV genome in SCC (Rutten et al., 1992; Fornazari et al., 2017). However, Karakurt et al., (2023) reported two positive cases in which one case revealed homology with BPV-1. Low detection rate could be attributed to the limited sensitivity of PCR primers used. Future studies employing broader BPV typing strategies may provide a more comprehensive assessment.

Fig 7: PCR amplification of mammalian IGF-1 gene.



Fig 8: PCR amplification of L1 gene of Papillomavirus.

CONCLUSION

The present study demonstrated higher occurrence in cattle, particularly in crossbred Holstein-Friesian and aged female. Immunohistochemistry confirmed strong expression of PCK, PCNA, p120, p63 and γ-H2AX, highlighting their diagnostic and biological significance. PCR confirmed the presence of Papillomavirus in bovine SCC. However, future studies using broader BPV typing approaches are warranted.

ACKNOWLEDGEMENT

All authors are thankful to the authorities of Kamdhenu University and The Principal, COVSAH, KU, Sardarkrushinagar for providing all the resources. Also thank to Dr. Harshal Solanki and Dr.K.N.Panchal for providing support in sample collection.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The research protocol was approved by the Institutional Animal Ethics Committee (IAEC), College of Veterinary Science and Animal Husbandry (COVSAH), KU, Sardarkrushinagar, Gujarat, vide letter no. VETCOLL/IAEC/2025/24/PROTOCOL-4.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Patho-epidemiological and Immunohistochemical Study of Squamous Cell Carcinoma in Cattle and Buffaloes

    R
    Riddhi V. Trambadiya1
    0009-0009-5430-5479
    J
    Jignesh M. Patel1,*
    S
    Samir H. Raval2
    R
    Rohit S. Parmar3
    J
    Jignesh B. Patel4
    1Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar- 385 506, Gujarat, India.
    2Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand-388 001, Gujarat, India.
    3Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari-396 450, Gujarat, India.
    4Department of Veterinary Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.

    ABSTRACT

    Background: Study was conducted to investigate the etiopathogenesis of squamous cell carcinoma (SCC) in cattle and buffalo through pathoepidemiological, histopathological, immunohistochemical and molecular analysis.

    Methods: A total of 64 tissue samples of SCC were collected between December 2024 and December 2025.  Immuno histo chemistry (IHC) was performed to detect immunoexpression of pan cyto keratin (PCK), proliferating cell nuclear antigen (PCNA), zonula occludens-1 (ZO-1), E-cadherin, Vimentin, FOXA1, p120, GATA3, γ-H2AX and p63. Polymerase chain reaction (PCR) was performed to detect papillomavirus.

    Result: Out of 64 cases, ocular tumors were the most prevalent, followed by horn cancer (34.38%) and vulvar tumors (9.37%). Cattle accounted for 87.5% cases, with crossbred Holstein-Friesian showing the highest occurrence. SCC was most frequently observed in females and in animals aged 7-10 years. Histopathologically, most tumors were well differentiated (WD) (70.31%). Immunohistochemically, SCCs exhibited strong expression of PCK, PCNA, p120, γ-H2AX and p63, while ZO-1 showed moderate expression. Upregulation of vimentin and downregulation of E-cadherin indicated epithelial-mesenchymal transition (EMT), whereas increased γ-H2AX expression suggested enhanced DNA double-strand damage in poorly differentiated tumors. PCR confirmed the presence of papillomavirus in four (6.25%) SCC cases.

     

    INTRODUCTION

    Cutaneous tumors are the most frequently diagnosed neoplasms in veterinary medicine (Goldschmidt and Goldschmidt, 2016). SCC is the most common malignant epithelial neoplasm in livestock and can arise at multiple anatomical sites viz. eye, horn, vulva, teat, udder, perineum and skin. In India, SCC accounts for approximately 80% of reported neoplasms in cattle and 3% in buffaloes (Somvanshi, 1991). Several risk factors have been implicated in the development of the tumor, such as chronic ultraviolet (UV) radiation, non-pigmented skin, lack of hair, chronic irritation, trauma, breed predisposition, age and sex (Velavan et al., 2025).
           
    Immunohistochemistry was performed using a panel of biomarkers to evaluate different biological aspects of SCC, including PCK for epithelial origin, PCNA for cellular proliferation, ZO-1, E-cadherin and p120-catenin for cell adhesion and epithelial integrity, vimentin for EMT, γ-H2AX for DNA double-strand break, p63 for squamous differentiation and FOXA1 and GATA3 as transcription factors (Mestrinho et al., 2015; Karakurt et al., 2021). A limited number of studies have evaluated immunohistochemical biomarkers associated with proliferation, EMT and genomic instability in SCC in a single cohort.
           
    Papillomaviruses are strongly associated with papillomas and SCCs across multiple species. However, the exact mechanisms involved in carcinogenesis remain poorly understood. This limits the understanding of tumor behavior, progression and diagnostic biomarker utility in farm animals.
           
    The objectives of the present study were to investigate the pathoepidemiological and pathomorphological characteristics of SCC in cattle and buffaloes, characterize histopathological lesions, evaluate immunoexpression of selected biomarkers and determine the presence of papillomavirus using PCR.

    MATERIALS AND METHODS

    Study period and location
     
    The research work was conducted at the Department of Veterinary Pathology, COVSAH, KU, Sardarkrushinagar, Gujarat between December, 2024 and December, 2025.
     
    Sample collection
     
    A total of 87 suspected samples were examined. Histopathological evaluation confirmed 64 cases as SCC, while 23 tissue growths were diagnosed as non-neoplastic and excluded from further analyses. Among 64 cases, 22 horn cancer (HC), 36 ocular SCC (OSCC) and 6 vulvar SCCs (VSCC) were noted (Supplementary Material 1). The tissue specimens were collected from 1) Veterinary Clinical Complex, COVSAH, KU, Sardarkrushinagar 2) Anchorwala Ahinsadham, Mundra and 3) Jalaram Gaushala, Bhabhar. Tissue was collected in 10% neutral buffered formalin (NBF) for histopathological analysis and in sterile containers for molecular analysis without any preservatives and stored at -20°C as soon as possible.

    Supplementary Material 1: Details of samples collected (n=64).


     
    Histopathology
     
    All collected tissues were fixed in 10% NBF, subjected to standard processing. The entire tissue processing was accomplished with automatic tissue processor and subjected to hematoxylin and eosin (HandE) staining according to standard protocol of Luna (1968). SCCs were graded according to the Modified Anneroth’s grading system (Table 1).

    Table 1: Modified anneroth’s grading system.


     
    Immuno histo chemistry (IHC)
     
    Histopathologically confirmed slides were subjected to immunohistochemical examination with various biomarkers using standard protocol of Raval et al., (2018). The information regarding primary antibody used in this study were mentioned in Table 2.  After TBS rinsing, sections were incubated with Envision rabbit/mouse reagent conjugated with peroxidase (Dako, Denmark) for 30 minutes. Chromogenic reaction was visualized using 3,3’-diaminobenzidine (DAB) and counterstained with Harris-hematoxylin, dehydrated, cleared and mounted.

    Table 2: Details of Primary antibody used in IHC.


           
    Immunohistochemical scoring was carried out by a semi-quantitative scoring system by assessing the percent of positive immunoreactive cells i.e. for PCK (Baghla et al., 2012; Kumar et al., 2025), for PCNA (Kumaraguruparan et al., 2006), for ZO-1 (Upadhaya et al., 2022), for E-cadherin (Pinho et al., 2007), for p120 (Rajeev et al., 2024), for γ-H2AX (Dörtbudak and Yener, 2025) and for p63 (Mestrinho et al., 2015). Strong nuclear immunoreactivity for FOXA1 and strong nuclear staining in ≥10% neoplastic cells were considered as positive to GATA3 biomarker (Machado et al., 2020).
     
    Molecular detection of oncogenic virus
     
    DNA extraction
     
    The DNA isolation from tissues was accomplished using the commercially available DNeasy Blood and Tissue Kit manufactured by QIAGEN-GmbH, Germany according to the manufacturer’s instructions. All DNA was subjected to quality evaluation using highly conserved mammalian IGF-1 gene (Table 3).

    Table 3: Specifications of primers.


     
    Detection of Papillomavirus
     
    Primer specific to papillomavirus were adopted from Forslund et al., (1999) (Table 3). Reaction mixture was prepared containing 10 μL of PCR mastermix, 0.8 μL of each primer, 2.4 μL template DNA and 6 μL PCR grade water. PCR was performed in thermal cycler (The Veriti® Applied Biosystems) according to protocol mentioned in Table 4. The detection of amplified products were performed in 1.5% Agarose gel at 85 voltages for 40 minutes and the DNA bands were visualized using Dual LED Blue/White Light Transilluminator and photographed.

    Table 4: Protocol for PCR.

    RESULTS AND DISCUSSION

    Incidence
     
    Current study reported that OSCC was the predominant tumour, followed by HC and VSCC (Table 5) and found similarity with an earlier study (Parmar et al., 2024). In cattle 56 cases were recorded while in buffaloes only 8 cases were found, that were similar to the findings revealed by Reddy et al., (2025). Among HC, 19(86.36%) were noticed in cattle whereas 3(13.64%) were diagnosed in buffaloes. Similarly OSCC and VSCC were reported higher in cattle as compared to buffaloes.

    Table 5: Histopathological grading of SCC.


           
    Highest incidence was noted in HF-crossbred cattle (44.64%) followed by Kankrej (25.00%), Non-descript (21.43%) and Gir (8.93%); found compatible with Yeruham et al., (1999) and Parmar et al., (2024). In buffaloes, equal distribution was reported among Mehsana(50%) and Banni breed(50%).
           
    Highest incidence was observed in the 7-10 years age group(n=47), followed by <7 years(n=13) and the least in >10 years(n=4), which was comparable to Dave et al., (2025). However, Udharwar et al., (2008) reported higher occurrence in >10 years. There was equal incidence of HC among male and female. Higher occurrence of bovine OSCC was noted in females (86.11%) and were in agreement with Kalim et al., (2021) and Nithya et al., (2022).

    Gross pathology of SCC
     
    Gross pathomorphology of affected parts were recorded (Supplementary Material 2). In HC, majority cases (n=12) exhibited cauliflower shaped growth at base of affected horn; while others showed nodular growth with verrucous surface (n=10). Most growths had a firm consistency. Some large ulcerated growth showed bleeding from wound. Cut sections revealed yellowish-white, grayish-white, reddish-brown to brownish-black surface along with widespread hemorrhage and necrosis (Desai et al., 2024; Kumar et al., 2025).

    Supplementary Material 2: Gross pathomorphological features of suspected cases of SCCs.


           
    Majority OSCC (n=19) were found on nictitating membrane followed by lower eyelid, bulbar conjunctiva, corneo-scleral junction and upper eyelid, respectively. Majority had cauliflower like appearance followed by nodular growth with rough and verrucous surface. Few cases had papillomatous and pedunculated growth. Similar alterations were revealed by Fornazari et al., (2017), in contrast to Karakurt et al., (2023).
     
    Histopathology of SCC
     
    Histopathological grading was carried out and divided into WD, MD and PD tumors (Table 5). Scoring of individual tumorous mass was noted (Supplementary Material 3).

    Supplementary Material 3: Histopathological scoring of individual SCC as per modified Anneroth’s grading system.


           
    Histopathology of WD-HC revealed proliferating neoplastic cells forming concentric lamellae of keratins developing large epithelial pearls (Fig 1A). Prominent keratinization was observed in center of islands (Joshi et al., 2009). Cells were arranged in islands and showed moderate pleomorphism. One case showed a transition between pseudostratified columnar epithelium to SCC (Fig 1B) and also showed interconnected osseous trabeculae that supported by loose fibrovascular stroma (Fig 1C). Mitotic index was very low. MD neoplasms revealed neoplastic cells arranged in cords and trabeculae and showed pleomorphism and apoptosis.

    Fig 1: Histopathology.


           
    Well-differentiated OSCC revealed presence of round to polygonal shaped cells arranged in islands, cords and trabeculae, supported by fibrovascular stroma and typical keratin pearls (Fig 1D). Neoplastic cells had distinct cell borders and showed anisocytosis and anisokaryosis, karyomegaly, apoptosis and few mitotic figures (Fig 2A). Few cases revealed marked granulomatous inflammation characterized by infiltration of macrophages, multinucleated giant cells, plasma cells, lymphocytes and polymorphs cells (Fig 2B). WD and MD neoplasms showed multifocal neoplastic cell with prominent intercellular bridges (desmosomes) (Fig 2C). PD infiltrative neoplasm showed polygonal cells arranged in cords and trabeculae. Mononuclear cells infiltration and atypical mitotic figures were noted (Sharma et al., 2020; Karakurt et al., 2023).

    Fig 2: Histopathology.


           
    VSCC showed proliferating neoplastic cells arranged in small islands that formed concentric layers, developing multiple prominent characteristic epithelial pearls (Fig 2D). Neoplastic cells showed minimal anisocytosis and anisokaryosis (Khodakaram-Tafti et al., 2013).
     
    Immuno histo chemistry of SCC
     
    Immunohistochemical examination was performed using aforementioned various tumor markers. IHC revealed all cases of SCCs (100%) were positive for PCK, PCNA and p63; 42 (65.63%) for ZO-1; 35 (54.68%) for E-cadherin; 39 (60.94%) for p120; 52 (81.25%) for Vimentin; and 56 (87.50%) were for γ-H2AX. However, FOXA1 and GATA3 failed to react with any tumorous tissue. Immunohistochemical scoring of individual tumor mass for each marker is given in Supplementary Material 4.

    Supplementary Material 4: Immunohistochemical scoring of individual SCC.


           
    Current study reported that all cases showed cytoplasmic immunoreactivity to PCK. Out of 64, 4(6.25%) cases depicted immunoreactivity in >75% neoplastic cells and were considered strongly immunoreactive (Fig 3A). However, 25(39.06%) cases showed expression in 51-75% cells, 24(37.50%) in 26-50% cells and 11(17.19%) in <25% cells. WD-SCC showed strong cytoplasmic immunoreactivity with pancytokeratin in neoplastic cells and keratin pearls (Fig 3B). The present findings were similar to those of Kumar et al. (2023). The strong immunolabelling of PCK indicates tumor of epithelial origin.

    Fig 3: Immunohistochemistry.



    Among 64 cases, 7 had nuclear immunoreactivity to PCNA in >75% of neoplastic cells and were considered strongly immunoreactive. However, 26 cases showed expression in 51-75% cells, 28 showed expression in 26-50% cells and 3 showed expression in 5-25% of cells. These findings suggested that WD-SCC showed mild to moderate nuclear immunostaining (Fig 3C), while PD-SCC showed strong immnunostaining in proliferating neoplastic cells (Fig 3D) that suggests proliferative potential and biological aggressiveness of the tumor. These findings were in accordance with Martano et al., (2016). However, Karakurt et al., (2021) did not find any significant difference. 

           
    Among 64 cases, only one case (1.56%) showed membranous immunostaining of ZO-1 in >75% neoplastic cells, 13(20.31%) in ≥50-<75% cells, 19(29.69%) in ≥25-<50 cells, 9(14.06%) in ≥10-<25 cells and 22(34.38%) were in <10% neoplastic cells. 54 cases showed weak immunostaining (Fig 4A), while 10 had moderate membranous staining (Fig 4B). All tumors exhibited low ZO-1 expression in current study (Qiu et al., 2016; Upadhaya et al., 2022), indicating disruption of tight junction integrity during malignant transformation.

    Fig 4: Immunohistochemistry.


           
    Out of 64 SCCs, 35(54.68%) cases showed positive immunoreactivity with E-cadherin (Fig 4C) and 29 were found non-immunoreactive. Among the 35 positively expressed tumors, 20 cases (20/35; 57.14%) showed immunolabelling in <25% neoplastic cells and 15(42.86%) cases showed in 26-50% neoplastic cells. Upadhaya et al., (2022) reported complete disruption of the adherens molecule in all cases. This down-regulation of E-cadherin indicates disruption of adherens junctions in epithelial tissues as cells progresses towards malignancy.
           
    Among 64 SCCs, 52(81.25%) cases were found immunopositive for vimentin (Fig 4D), while 12 cases were found non-immunoreactive. This suggests that EMT is crucial for tumor invasion and metastasis. The present finding was found compatible with Yu et al., (2015).
           
    FOXA1 did not show any nuclear immunoreactivity in neoplastic cells and was thus considered as non-immunoreactive (Fig 5A). This negative immunoreactivity could be due to the antibody clone failing to react with bovine tissue. Similarly, all cases were found negative for GATA3 immunoreactivity (Fig 5B). Liu et al., (2012) also reported similar findings in SCC cases.

    Fig 5: Immunohistochemistry.


           
    A total of 39(60.94%) cases showed p120 membranous expression in >50% neoplastic cells and were considered to show preserved expression of p120 (Fig 5C). Whereas, 25(39.06%) cases showed <50% of positive neoplastic cells and considered as reduced expression (Fig 5D). Li et al., (2020) also reported decreased expression in SCC, which may indicate loss of cell adhesion and enhanced invasive potential of neoplastic cells.
           
    In this study, 56 (87.50%) were found immunoreactive for γ-H2AX whereas 8 (12.50%) were found negative. Out of 56, 31(55.36%) cases showed mild nuclear immunoreactivity (Fig 6A), 16(28.57%) showed moderate immunoreactivity and 9(16.07%) showed severe immunoreactivity (Fig 6B). While 8(12.50%) cases were found non-immunoreactive similar to Dörtbudak and Yener (2025). Chronic ultraviolet (UV) radiation exposure is considered an important etiological factor that can induce DNA damage, leading to increased γ-H2AX expression.

    Fig 6: Immunohistochemistry.


           
    Furthermore, all cases were found immunoreactive for p63 (Fig 6C) and (Fig 6D). A total of 15(23.44%) cases showed p63 expression in >70% neoplastic cells, 24(37.50%) cases in >50-<75% cells and 25(39.06%) cases in <25% neoplastic cells. In current study, p63 expression showed a significant correlation with the degree of tumor differentiation similar to Mestrinho et al., (2015).

    Molecular detection of oncogenic virus
     
    All extracted DNA tested positive on the quality-assessment using PCR for IGF-1 gene (Fig 7). Present investigation reported successful molecular detection of Papillomavirus in only four cases (6.25%) (Fig 8). Earlier studies were unable to detect BPV genome in SCC (Rutten et al., 1992; Fornazari et al., 2017). However, Karakurt et al., (2023) reported two positive cases in which one case revealed homology with BPV-1. Low detection rate could be attributed to the limited sensitivity of PCR primers used. Future studies employing broader BPV typing strategies may provide a more comprehensive assessment.

    Fig 7: PCR amplification of mammalian IGF-1 gene.



    Fig 8: PCR amplification of L1 gene of Papillomavirus.

    CONCLUSION

    The present study demonstrated higher occurrence in cattle, particularly in crossbred Holstein-Friesian and aged female. Immunohistochemistry confirmed strong expression of PCK, PCNA, p120, p63 and γ-H2AX, highlighting their diagnostic and biological significance. PCR confirmed the presence of Papillomavirus in bovine SCC. However, future studies using broader BPV typing approaches are warranted.

    ACKNOWLEDGEMENT

    All authors are thankful to the authorities of Kamdhenu University and The Principal, COVSAH, KU, Sardarkrushinagar for providing all the resources. Also thank to Dr. Harshal Solanki and Dr.K.N.Panchal for providing support in sample collection.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    The research protocol was approved by the Institutional Animal Ethics Committee (IAEC), College of Veterinary Science and Animal Husbandry (COVSAH), KU, Sardarkrushinagar, Gujarat, vide letter no. VETCOLL/IAEC/2025/24/PROTOCOL-4.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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