Molecular Diagnosis and Application of Combined Alternative Treatment in Lesions Developing in the Oral Region due to Orf Virus in Sheep and Goats

Orf virus (ORFV) is a zoonotic pathogen that infects sheep and goats and is responsible for significant economic losses (Karki et al., 2019). ORFV belongs to the Parapoxvirus genus, Chordopoxvirinae subfamily and Poxviridae family (Cargnelutti et al., 2011). Bovine papularstomatitis virus (BPSV) and Pseudocowpox virus (PCPV) are the most important members of this family (Cargnelutti et al., 2011; Khalafalla et al., 2020). ORFV enters the live body through damaged tissues and replicates in epidermal cells (Atli, 2017; Bergqvist et al., 2017). It usually spreads to healthy animals through contact with infected animals. ORFV infection in sheep and goats causes proliferative and self-limiting crustal lesions that can be seen frequently on the skin of the lips, around the nostrils and rarely on the feet and teats (Cargnelutti et al., 2011; Fleming et al., 2015). ORFV infection is widespread worldwide and endemic in countries where sheep and goats are raised. While ORFV infection in sheep and goats has low mortality, it has a mortality rate of 10% to 90% in lambs and kids due to their failure to suckle their mothers and secondary bacterial infections (Nandi et al., 2011; Atli, 2017; Karki et al., 2019). Vaccines (live or live attenuated) are commonly used for the prevention of ecthyma disease caused by ORFV in sheep and goats (Karki et al., 2019). The need for new approaches in the treatment of this disease has arisen due to the recent occurrence of the disease throughout the year, which was previously seen seasonally in animals. In this study, the aim was to determine the presence of viral genomes in lesions developed in the mouths (lips, gums) of sheep and goats due to ORFV using specific ORFV primers and to investigate the effectiveness of triple combined alternative treatment methods in these animals.

Samples were collected from the lesions on the lips and gums of 29 sheep and 29 goats aged 2 years and older with ORFV infection from various sheepfolds in Burdur-Merkez and its districts. All samples were collected, all field tests were performed and all analyses were carried out in our laboratories (Departments of Virology and Pathology, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University) between January and April 2023. DNA extraction was performed from the lesion crusts using the DNeasy Blood and Tissue Kit (Qiagen, Germany). The application was carried out according to the protocol provided by the manufacturer. The obtained DNA extract was stored at -20°C until PCR application. In PCR applications, typing of ORFV in ecthyma samples collected from sheep and goats in Burdur province was performed using 7 different types of primers from Metabion (Synthesis Report Metabion International AG, Germany). For this purpose, the primers used in this study (GIF/IL-2, PPP1-PPP4, Orf1-Orf2, VIR1-VIR2, vIL-10-3 vIL-10-4, B2L and Alpha tubulin) were applied by modifying the procedures developed by different researchers (Inoshima et al., 2000; Kottaridi et al., 2006a; Gu et al., 2011; Alam et al., 2012; Mwanandota et al., 2016; Adedeji et al., 2017). The amplified products were then run on 1.5% Tris Acetate EDTA (TAE) agarose gel with 10 μl safe view dye (ABM, Canada) using a thermal cycler device and visualized using an imaging system (BIO-RAD GelDoc Go Imaging System, USA). Samples from oral mucosa lesions were collected and fixed in 10% neutral formalin solution. The tissue samples were then processed using Leica ASP300S (Leica Biosystems, USA) fully automatic tissue processing equipment and embedded in paraffin wax. Subsequently, 5 µm thick sections were obtained from the paraffin blocks using a Leica RM2155 rotary microtome (Leica Biosystems, USA) after cooling. These sections were stained with hematoxylin-eosin (HE) and examined under a light microscope. The sections were placed on polylysine slides and stained with streptavidin biotin peroxidase for immunohistochemical analysis. For ORFV detection, sheep orf antibody (positive control) was used as the primary antibody [sheep orf virus disease antibody IgG (ORFV-IgG) ELISA Kit, SL00097Sp, Sunlong Biotech Co. Ltd., China]. DAB chromogen and the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ab64264) were used as secondary antibody kit. After counterstaining the prepared slides with Harris Hematoxylin, a cover slip was applied and the slides were then examined under a microscope. A triple combination therapy was tried in all animals in which the presence of ORFV viral genome was investigated and positively detected; the positive cases were treated daily with 2 to 5 grams of PAPILEND^TM,^®cream (Almer Kimya İlaç San. Tic. Ltd.Şti., Ankara, Turkey) according to severity of lesions for 10 days, 2 doses of subcutaneous injection of 1 mL per 50 kg live weight of Ivermectin (Zolimectin^TM, Zoleant İlaç A.Ş. Şiþli, İstanbul, Turkey) at 15-day intervals and daily 10 grams of Alquermold^TM premix powder (Biovet, S.A., 25 Poligono Industrial, Tarragona, Spain) per animal for 10 days was added to animal feed.

PCR Results
 
In the study, with the use of 7 different types of primers as previously detected intensively in our region (Kaya and Kale, 2022), the presence of viral genome was detected in 10 out of 29 sheep (34.48%) and 14 out of 29 goats (48.28%) (Table 1). The GIF/IL-2 (2), PPP1-PPP4 (2) and B2L (2) primers were detected the most in sheep, while Orf1-Orf2 (4) and B2L (2) primers were detected the most in goats (Table 1, Fig 1).
 

 

 
Histopathology and Immunohistochemistry
 
According to histopathological analyses, acanthosis was caused by epidermal hyperplasia, hyperkeratosis and parakeratosis, as well as pronounced thickening of the epidermis. In addition, there was degeneration and necrosis of the epithelial cells, along with the presence of multifocal or consolidating necrotic crust on the epidermal layer. The most common finding was the marked epidermal extensions of the epidermis into the dermis, which are called rete ridges. Increased mitotic activity was noticed in epidermal cells. Degenerative and necrotic changes in the stratum spinosum’s spinous cells were also evident, with spongiosis, vacuolation, pyknotic and karyorrhectic nuclei. Micro abscess, scab development and peripheralized keratohyalin granules were common in the lesions. Keratinocytes exhibited typical intraepithelial ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies. Inflammatory cell infiltration, intracellular edema and sporadic hemorrhages caused the superficial dermis to enlarge. At the immunohistochemical examination, positive reactions with ORFV were detected in both epidermal and dermal cells (Fig 2).
 

 
Results of alternative treatment application
 
Successful (complete healing or regression) results were obtained in the oral lesions developed as a result of the triple combination application in all animals with positive detection of ORFV viral genome (Fig 3). The complete healing or regression time in sheep and goats with positive detection of ORFV viral genome and developing oral lesions occurred between 5-21 days as a result of the triple combination applications.
 

       
The most important way to achieve economic gain in sheep and goat herds is to obtain healthy offspring and increase the herd size. ORFV infection in sheep and goats commonly causes proliferative and self-limiting scab lesions on the lip skin, around the nostrils, rarely on the oral mucosa, feet and teats (Atli, 2017).
       
In the study, using 7 different types of primers that we previously detected intensively in our region (Kaya and Kale, 2022), viral genome presence was detected in 10 out of 29 sheep (34.48%) and 14 out of 29 goats (48.28%). Tedla et al., (2018) determined the ORFV prevalence in sheep as 15.5% and in goats as 8.5%, while Ifende et al., (2019) reported the ORFV prevalence in sheep as 4.2% and in goats as 3.6%. In contrast, Esmaeili et al., (2021) obtained a result of 44.6% positive for ORFV presence in sheep and 45.5% in goats.
       
In this study, sheep had the highest detection of GIF/IL-2, PPP1-PPP4 and B2L primers, whereas goats had the highest detection of Orf1-Orf2 and B2L primers. Alam et al., (2012) and Kaya and Kale (2022) have previously identified the presence of ORFV GIF/IL-2 primers in sheep and goats and in lambs and kids, respectively. PCR studies using PPP1-PPP4 primers in sheep and goats have been found positive in some studies (Kottaridi et al., 2006b; Castro et al., 2019; Şevik, 2019; Esmaeili et al., 2021). In a study conducted in Nigeria, 100% positivity was found in 60 male goats using Orf1-Orf2 primers (Adedeji et al., 2017). B2L gene primers were found in 34 adult local breed goats from Belbis and Sharkia cities in northern Egypt by Shehata et al., (2019). Zeedan et al., (2015) detected positivity in 3 out of 15 sheep and 4 out of 15 goats using B2L primers in a PCR analysis, while El-Tholoth et al. (2015) found 24 out of 30 PCR-positive results using B2L primers in a study conducted on a farm with 500 animals showing clinical signs of ORFV. Our study also identified positivity using VIR1 VIR2, vIL-10-3 vIL-10-4 and Alpha-tubulin primers. Indeed, the presence of VIR1 VIR2 (Maganga et al., 2016), vIL-10-3 vIL-10-4 (Kottaridi et al., 2006b) and Alpha-tubulin (Markoulatos et al., 2000; Kottaridi et al., 2006b) has been identified in sheep and goats by various researchers.  
       
The infected animals had the typical multifocal to consolidating, ulcerated lesions on the epidermis of the gums, lips, mouth margin, muzzles, nose and udder. Additionally, considerable proliferative papules, pustules, crusting and brownish scabs appeared on infected animals. Within a few days, orf pustules appear, followed in a few weeks by ulcers and a thick scab with no formation of scar tissue (Abbas and Mughal, 2014). A superficial lesion’s etiological agent may cause it to crust, scab over and eventually fall off. Additionally, opportunistic microbes could enter these wounds and spread disease (Kumar et al., 2014). The hallmarks of microscopic lesions in the vesicular stage include marked epidermal hyperplasia, ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies within keratinocytes (Tahir et al., 2014; Vellucci et al., 2020). Additionally, the stratum granulosum and spinosum are the main locations for keratohyalin granules. These granules, which are water-insoluble and support cell dehydration, are present in the cytoplasm. These granules go through cellular adjustments during keratinocyte development that lead to the conversion of keratin tonofilaments into a homogenous keratin matrix, a crucial step in cornification (Hermanns-Le et al., 2004; Takahashi et al., 2010). The macroscopic and histopathological lesions observed in this study were in consistent with ORFV infection in sheep and goats, according to earlier research (Ozmen and Dolu, 2018; Aneed and Al-Saad, 2019; Hussain et al., 2022).  
       
Various alternative methods have been applied for the treatment of contagious ecthyma disease in sheep and goats caused by ORFV (Geerlings and Wageningen, 2001; Liu et al., 2006; Sunitha et al., 2019; Wang et al., 2019) and successful results have been achieved in these studies. In this study, successful healing / regression was observed in the oral lesions that developed as a result of the triple combination application in all animals with positive ORFV viral genome. It was determined that the complete healing or regression period occurred within a range of 5-21 days as a result of triple combination applications. In this study, topical PAPILEND^TM® cream was applied daily to the lesions that developed as a result of the triple combination application in all animals with positive ORFV viral genome presence. The PAPILEND^TM® cream product contains Glacial acetic acid, Salicylic acid, Garlic oil, Tea tree oil, Glycerol monostearate, Stearic acid, Cetyl stearyl alcohol, Hydrogenated castor oil, podophyllin and water. Some of the ingredients in PAPILEND^TM® cream show topical cytotoxic and antimitotic effects. It has been reported that some of these ingredients (e.g. Tea tree oil, podophyllin) stop mitosis in epithelial cells at the metaphase stage, are very effective on warts with less keratin deposition such as plantar warts compared to warts with intense keratin deposition, have lytic effects that do not extend beyond epidermal cells, remain intact in the basal layer, do not cause scar formation when topically applied, are absorbed systemically and when applied to bleeding or recently biopsied warts, the amount of absorption increases (Saltýk et al., 2022). In addition to these animals, 1 mL of ivermectin was administered subcutaneously every 15 days to animals weighing 50 kg, for 2 times. Ivermectin is a potent antiparasitic drug that is effective against many helminths and arthropods and belongs to the macrocyclic lactone class. Avermectin B is derived from Streptomyces avermitis, an actinomycete found in soil. While structurally similar to macrolides, it does not exhibit antibacterial activity (Omura, 2008).
       
Ivermectin stimulates both cellular and humoral immunity. The prevalence of papillomas in the body is linked to the immune system. Therefore, cellular immunity is more effective than humoral immunity in reducing papillomas. Ivermectin’s anti-tumor activity has also been reported. In mice, ivermectin application has been shown to increase antibody production, serum specific antibody activity and responses from T lymphocytes and macrophages (Rao et al., 1987; Blakley and Rousseaux, 1991; Uhlir, 1991; Drinyaev et al., 2004; Korystov et al., 2004; Börkü et al., 2007). Additionally, in the study, infected animals were given 10 grams of AlquermoldTM premix powder per day per animal for 10 days. Vitamin E, selenium, copper and zinc are important substances that strengthen the immune system against diseases in cattle (Spears and Weiss, 2008). Vitamin E is mainly obtained from feed (Baldi et al., 2000). When prepared feed is stored for a long time or processed, the amount of vitamin E decreases. Additionally, when grazing, they may not receive enough vitamin E from plants (Baldi, 2005). Selenium may not be present in sufficient amounts in the soil or in plants (Sharma et al., 1983). Selenium is an essential component of the organism’s antioxidant defense system (Kieliszek and Błażejak, 2016). Selenium exhibits a synergistic effect with vitamin E (Willshire and Payne, 2011). Zinc plays an important role in keratin production (Tomlinson et al., 2004). Copper and zinc are important in fighting microorganisms and strengthening the immune system (Spears, 2000).
       
Lyophilized live vaccines are widely used for protective purposes in Turkey and worldwide in cases of orf disease (Rziha et al., 2000; Musser et al., 2008; Hosamani et al., 2009; Bimeda Biologicals, 2022; Colorado Serum Company, 2022; Dollvet, 2022; NOAH Compendium, 2022; Ministry of Agriculture and Forestry Istanbul Pendik Veterinary Control Institute, 2022; Vetal, 2022). Advances in the field of recombinant DNA vaccines are still ongoing for the development of ORFV vaccines through experimental studies (Orta et al., 2022). Vaccination is the most basic protection method against orf disease, but short-term immunity and the need for annual revaccination are significant drawbacks. Therefore, alternative treatment methods can be used in the early stages of the disease to prevent its spread within the herd. In recent years, the use of alternative treatment products developed by various researchers for orf disease has gained importance. Therefore, it has been suggested that efforts should be made to develop new commercial products in this field as well (Orta et al., 2022).

In conclusion, different ORFV primers and PCR test applications that are commonly used for detection can be appropriate and it is recommended to work with other different ORFV primers. Tests such as histopathology and immunohistochemistry can also be used for confirmation. In addition to ORFV preventive vaccinations in sheep and goats, our study has shown that the use of triple combined alternative applications was successful for treating affected animals effectively.