Neoplasms In Equine Species In Sharkia Governorate, Egypt

| Few studies and very limited published data concerning the spread of neoplasia among Equidae in Egypt are available, therefore, the present study was carried out to investigate the prevalence and types of neoplasia among Equidae in Sharkia Governorate, Egypt, and to assess the efficacy of some available biomarkers as a diagnostic tools. This work was investigated 35 clinical cases of equine neoplasms (23 donkeys, 10 horses, and 2 mules) out of 1513 total examined cases admitted from different clinics in Sharkia Governorate, from May 2019 to October 2020. Depending upon gross and histopathological findings; the neoplastic growths include 12 cases of sarcoids, 9 cases of fibroma, 4 cases of squamous cell carcinoma, 3 cases of papilloma, 2 cases of fibropapilloma, 1 case of fibrosarcoma,1 case of myxoma, 1 case of of myxosarcoma, 1 case of myxoid liposarcoma, and 1 case of ovarian teratoma. Immunohistochemically, cytokeratin5 (CK5), MDM2, and S100 biomarkers were used to confirm the diagnosis of certain neoplasms. The Overall prevalence of equine neoplasms was 2.31 %. The diagnosed cases were linked with animal species and sex and the significance (p < 0.05) of differences among age, seasons, and location of the neoplasms were evaluated. The prevalence rates in domestic donkeys, horses, and mules were 2.66 %, 1.94%and 1.22% respectively. The rate of affected animals over 3 years was (3.38 %) in comparison with animals less than 3 years (0.19 %). Females (2.64%) were more affected than males (2.28%) and mules (1.42%). The prevalence rate was 5.56% in summer, 1.6 % in spring, 1% in autumn, and 0.85 % in winter. A higher rate of neoplasms was reported in extremities and trunk (1.25%), 0.52% in the head, neck, 0.19% back, ventrum, 0.19% in eye, related skin, 0.13 % in genital organs.

November 2021 | Volume 9 | Issue 11 | Page 2006 Advances in Animal and Veterinary Sciences noma, hemangioma, basal cell tumor, adenoma, and mammary adenocarcinoma respectively constitute the remaining percentage of neoplasm cases (12.5%). Sarcoid was the most common in horses and was the only common tumor in donkeys and mules. While, ocular SCC was prevalent in paints and quarter horses, and penile SCC was most common in appaloosas and quarter horses.
In Egypt, there is little published data about the prevalence of equine neoplasms, but few surveys as by (Hassanein and Mahmoud, 2009) diagnosed the most common affected neoplasms of equine species in different localities of Egypt (Assiut, El-Menia, and Sohag) which were 32.1 % sarcoids and 1.48% fibroma in 202 tumors collected from different farm animals.
Sarcoid developed in head and neck (39%), extremities (35%), ventrum (26%), and any parts of the equine body that were susceptible to trauma (Knottenbelt and Kelly, 2000). The prepuce area, abdomen, flank, chest, and trunk together with wounds of castration and groins were also included (Carr et al., 2001). Lesions frequently occur at the sites of wounds or surgery as well as other areas that are frequently rubbed by horses to remove flies (Ghim et al., 2004).
Here we aimed to demonstrate the prevalence through morphological, histopathological studies and evaluate the efficacy of certain available biomarkers for neoplastic growths in addition to post-excisional follow up of some cases in Sharkia Governorate.

AnimAlS
Neoplastic growths were recorded in 35 equine species (23 donkeys, 10 horses, and 2 mules) among 1513 reared in different localities in Sharkia governorate from May 2019 to October 2020. A full description data for each case was recorded concerning their onset time, age, sex, region, shape, size, and consistency.

diAGnoSiS
All cases were subjected to clinical and histopathological examination. The clinical examination of the affected animals helped in the preliminary diagnosis as it gives information about the different clinical types of sarcoid which confirmed using the clinical classification of (Knottenbelt, 2005). Clinical evaluation was based on lesions appearance and reporting correlated skin lesions as ulceration, scaling, alopecia, and exudation.

hiStopAtholoGicAl exAminAtion
Total excisional biopsies were collected from all proliferative lesions. The tissue specimens were fixed in neutral buffered formalin 10% for 48 hours, dehydrated in ethyl alcohol, cleared in xylene, embedded in paraffin wax, sectioned into 5μm in thickness using automated microtome then stained with routine Hematoxylin and Eosin (H & E). Some special stains were applied as Masson's trichrome for staining collagen in fibroma cases. Moreover, an Alcian blue stain was used to demonstrate mucin in myxoid liposarcoma case. (Suvarana et al., 2018).
Paraffin sections (5 μm) were placed on charged slides, deparaffinized, hydrated then antigen retrieval was performed. After the sections were incubated with the primary antibodies overnight at 4 °C in a humidified chamber, slides were washed with phosphate-buffered saline (PBS) and incubated with the secondary antibody (Expose mouse and rabbit specific HRP/DAB detection kit, Abcam; Ready-to-use; Cat.#: ab80436) for 15 minutes at room temperature in a humidified chamber then rinsed with PBS. Staining was then performed using the DAB chromogenic agent for detection of immunoreactivity, counterstained with hematoxylin, dehydrated, and mounted in a synthetic medium.

StAtiSticAl AnAlySiS
All statistical operations were performed using SAS statistical analysis software system Package V9.1. Univariate logistic regression model was fitted through Maximum Likelihood (ML) procedure to assess the effect of independent variables [animal species, age, sex, season and locations of neoplasms], on the dependent dichotomous variable (prevalence of neoplasms). P-values of less than 0.05 are considered having a statistically significant association November 2021 | Volume 9 | Issue 11 | Page 2007 Advances in Animal and Veterinary Sciences

rESultS ANd dIScuSSIoN
Our survey identified of 35 clinical cases of equine neoplasms out of 1513 total examined cases. Summary about the age, sex, breed, location, and season of each collected case was presented in (Table 1).
Depending upon gross, histopathological and immunohistochemical findings; our results revealed 12 cases of sarcoids, 9 cases of fibroma, 4 cases of SCCs, 3 cases of papilloma, 2 cases of fibropapilloma, 1 case of fibrosarcoma,1 case of myxoma, 1 case of myxosarcoma, 1 case of myxoid liposarcoma, and 1 case of ovarian teratoma in mare. These results partially agree with (Valentine, 2006). (Pascoem and summers, 1981) found that the most commonly occurring neoplasms in horses were sarcoids, papillomas and SCCs of the eye and external genitalia. In another report by ( Jaglan et al., 2018) reported 4 cases of fibroma, one squamous cell carcinoma, and one papilloma in 6 clinical equine cases.
The detailed findings of recorded cases are described as follows: 1-Equine sarcoid: The most commonly recorded neoplasms in this study. According to the owners' complaints, the lesion appeared spontaneously and increase its size with time. The tumor appeared single or multiple firm or fleshy warts-like lesions or ulcerated growth which appeared grayish-white in color with some hemorrhagic spots. According to gross features, the majority of cases were fibroblastic type which manifested by fleshy ulcerated surface and serum exudation (Fig.1a). In contrast, the verrucous lesions are characterized by their warty appearance with variable degrees of flaking and scaling of their surfaces (Fig.1b).
Histopathological characteristics of equine sarcoid include epidermal acanthosis and hyperkeratosis with long rete ridges into the dermis (Fig.1c) that confirmed by (Scott and Miller, 2003). The dermal layer contains immature fibroblasts running in a wavy pattern and picket fence formation ( membrane) (Fig.1d) that agreed with (Hewes and Sullins, 2009;Semieka et al., 2012). The most epidermal layers were ulcerated with necrosis and hemorrhages and infiltrated with neutrophils. The superficial dermis showed thrombosis, hemorrhage, and inflammatory cells infiltration mainly neutrophils, lymphocytes, macrophages, and eosinophils.

2-Fibroma:
The fibroma cases appeared over the skin mainly on the periocular region (Fig.2a), at the medial aspect of the forearm (Fig.2b), forelimb and hind limbs, and ventral abdomen. The cases appeared grossly as pedunculated polypoid mass or wart-like nodule or large nodular spherical subcutaneous masses. All cases from donkeys were firm in consistency and grayish-white in color with a wet bleeding surface. The case located at the ventral abdomen had an ulcerated surface with necrotic debris and fleshy consistency. Histopathologically, the tissue section revealed a capsulated nodule with whirls of fibrous connective tissue which run in all directions with abundant collagen fibers in-between (Fig.2c). Masson's trichrome stain revealed a positive reaction of blue-greenish collagen fibers and dark brown nuclei (Fig.2d).

3-Squamous cell carcinoma (Scc):
The neoplastic masses were lobulated growth (Fig 3a), solid in consistency and yellowish red in color. Microscopically, the tissue sections showed exophytic and endophytic growths with the dermal neoplastic invasion of pleomorphic epithelial cells (Fig.3b) with or without keratinized pearl formation and obvious mitotic figures surrounded by inflammatory cells. (Fig.3c). Immunohistochemical reactions revealed cytoplasmic expressions within the epithelial forming keratin pearls by cytokeratin 5 (Fig.3d). Similar results obtained by (Chu and Weiss, 2002). Cytokeratin was expressed in case of tumor progression and carcinoma (Frohwitter et al., 2016).

4-Papilloma:
The cases were seated at head skin, caudal aspect at pastern joint (Fig.4a), and female genitalia. The lesions appeared as nodular grayish-white firm mass. Histopathologically, the tissue section revealed hyperkeratosis, acanthosis with finger-like papillae (Fig.4b) and spongiosis of the epidermal layers in addition to parakeratosis with remnant nuclei at the cornified layer.

6-Fibrosarcoma:
One case of fibrosarcoma located at the skin of the fetlock region. The mass appeared as a large firm nodule with a grayish red ulcerated surface (Fig.6a) and a whitish lobulated fleshy mass on the cut section. Microscopically, the growth was composed mainly of neoplastic fibroblasts that arranged in herringbone-like patterns with pleomorphic cells (Fig.6b), high mitotic index, angiogenesis, and tumor giant cells.

7-Myxoma:
One case of myxoma was seen in the skin of the neck (Fig.7a). The mass appeared as solid, raised grayish-white color with dark red hemorrhagic spots on cut sections. The neoplasm is characterized microscopically by capsule formation (Fig.7b) with different shapes of embryonic white fibroblasts with interlacing processes and secretions (Fig.7 c,d).

8-Myxosarcoma:
One case of myxosarcoma was found on the skin near to fetlock joint. The neoplastic mass was solid, slimy, and grayish-white color (Fig.8.a). The nodule exhibited microscopic characteristics of malignancy criteria, high mitotic index, giant cells (Fig.8b), hemorrhagic areas (Fig.8c), and cytoplasmic processes.

9-Myxoid liposarcoma:
Large glistening neoplastic mass at forelimb region with the slimy cut surface (Fig.9a), the content of the growth comprised pleomorphic cells which ranged from the spindle, polygonal, oval cells which embedded in a mucoid substance containing poorly differentiated fat cells with undetectable collagen contents and variable sizes giant cells (Fig.9b)   chemical reactions revealed positive nuclear immunostaining with MDM2 (Fig.9d) and S100. In which, the genetic abnormalities can lead to specific nuclear protein overexpression of MDM2 that was previously detected by (Avallone et al., 2016). (Doria-Torra et al., 2015) applied an S100 biomarker to rule out a fibrosarcoma and confirmation of liposarcoma which give positive nuclear and cyto plasmic expressions. 10-ovarian teratoma: In a 7-year-old mare with an enlarged firm yellowish rounded ovarian mass (Fig.10.a) with difficulty on cut sections. Microscopically, this mass is formed from tissues like cartilage, bone, and skin with ovarian tissue (Fig.10.b,c). Teratomas have been seen in the bitch, sow, mare, and cow and most of them are well-differentiated and benign ( Jubb et al., 2007). Moreover, (Hassanein and Mahmoud, 2009)    seen due to hormonal disturbances and leads to a reduction in reproductivity and economic losses.
Collectively, the macroscopic and microscopic pictures of all confirmed cases were recorded in (Table 2).

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All equine species were susceptible to neoplasms but the prevalence rate in donkeys (2.66 %) was higher than horses (1.94%) and mules (1.22%), the previous results come in agreement with (Yu, 2006). This could be due to exposure of donkeys to bad managemental conditions during drafting in different villages of Sharkia governorates. (Valentine, 2006) reported that the only common reported neoplasms in donkeys were sarcoids. Moreover, Quarter horses and Arabians have a high risk for sarcoid development. The higher incidence of sarcoid reflects higher exposure to bovine papillomavirus due to a large number of equine species were cohabited in places with bovine species with a higher density of predisposed animals or of viral vectors (Scott and Miller, 2003). Sarcoid neoplasms may cause discomfort due to continuous irritation and can result in ulceration, infection, and occasionally lameness associated with lesion location.
The prevalence rate was significant (p < 0.05) with age factor where equine over 3 years had prevalence rate (3.38%) higher than animals less than 3 years (0.19%).These might be due to a long time of exposure to different predisposing factors causing neoplasms. In the present study, the mean age of affected animals ranged from 6 to 8 years, although other studies found an incidence peak between 3 and 6 years of age (Knottenbelt, 2005). However, a gradual increase in incidence is observed up to the age of 15 years followed by a decrease in prevalence (Mohamed et al., 1992). Other researchers have reported a higher prevalence in younger individuals (Clottu, 2008).
The prevalence rate of diagnostic cases was linked with sex in which the prevalence rate of females (2.64%) was more than males (2.28%) and mules (1.42%). These could be attributed to exposed females to immunosuppressive agents as pregnancy, parturition, and lactation.
There was a significant association (p < 0.05) between seasonal variations and the prevalence of neoplasms, in which a high prevalence rate was detected in summer (5.56%) and lower rates in spring (1.6 %), autumn (1% ) and winter 0.85 %. Flies may play important roles in transmission of bovine papillomavirus and irritation of minor wounds to become sarcoids. This factor reflects the importance of fly control in sites where equids are living especially in summer seasons in Egypt farms.
Other studies by (Martens et al., 2001) reported the trunk and head are more affected than the extremities and (Zahra et al., 2019) who recorded most sarcoid lesions in the head region then forelimbs, hind limbs, belly, back, and perineal region. On the other hand (Foy et al., 2002) reported that common sites of sarcoid are lips, eyelids and ears. The possible explanation for the high prevalence of limbs, shoulders, head and neck lesions might be due to continuous injuries and irritation afflicted by bad harnesses or grooming tools.
The prevalence rates of recorded equine neoplasms were summarized in (Table 3).
In Conclusion, we present data on the morphological, histopathological features and the prevalence of equine neoplasms in sharkia governorates, Egypt and evaluate the efficacy of certain available biomarkers using immunohistochemistry technique in addition to post excisional follow up of some cases