Effect of a Topical Formulation on Infective Viral Load in Lambs Naturally Infected with Orf Virus

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Veterinary Medicine: Research and Reports Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Effect of a Topical Formulation on Infective Viral Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 Load in Lambs Naturally Infected with Orf Virus Delia Lacasta 1 Introduction: Orf is a highly contagious eruptive viral disease of the skin and mucosa of Ramses Reina 2 sheep and goats. Although vaccination with live or attenuated orf virus is the preferred Marta Ruiz de Arcaute 1 option for disease control, the vaccine is unavailable in many countries. Treatment of orf Luis Miguel Ferrer 1 lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). Alfredo Angel Benito 3 The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine Maria Teresa Tejedor 4 For personal use only. and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was devel­ Irache Echeverria 2 oped for pain relief in sheep undergoing surgical husbandry procedures in Australia. Hector Ruiz 1 Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and Silvia Martinez Cardenas 1 buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It Peter Andrew Windsor 5 was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control. 1 Animal Pathology Department, Instituto Methods: One-month-old lambs (n=14), naturally infected with orf, were recruited from Agroalimentario de Aragón-IA2 a farm during a natural outbreak of the disease. The animals

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bs (n=14), naturally infected with orf, were recruited from Agroalimentario de Aragón-IA2 a farm during a natural outbreak of the disease. The animals were selected at the early stages (Universidad de Zaragoza-CITA), Veterinary Faculty of Zaragoza, Zaragoza, of the infection and randomly divided into two cohorts: Group A (n=11) treated with the 50013, Spain; 2Instituto de topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs Agrobiotecnología (CSIC-Gobierno de were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, Navarra), Mutilva, 31192, Navarra, Spain; 3 EXOPOL S.L, San Mateo de Gállego, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation Zaragoza, Spain; 4Anatomy, Embryology with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat and Animal Genetics Department, CIBER embryonic fibroblasts (TIGEF). CV (Universidad de Zaragoza-IIS), Veterinary Faculty of Zaragoza, Zaragoza, Results: Although no significant differences were found in the clinical progression of the 50013, Spain; 5Sydney School of lesions and PCR quantification (p=0.722) between these small cohorts, there was Veterinary Science, The University of Sydney, Camden, NSW, 2570, Australia a significant difference (p<0.05) in reduction in infective viral load between the groups when assessed in OSF cell cultures between T0 and T3. Conclusion: These preliminary findings suggest that treatment of early stage lesions with this TS may reduce the infective viral load present in orf lesions. Keywords: sheep, contagious ecthyma, wound formulation, local therapy Introduction Contagious ecthyma, also known as orf, contagious pustular dermatitis, sore mouth or scabby mouth, is a

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wound formulation, local therapy Introduction Contagious ecthyma, also known as orf, contagious pustular dermatitis, sore mouth or scabby mouth, is a highly contagious eruptive skin condition of sheep, goats and other ruminants, although zoonotic transmission also occurs. Orf disease affects mainly young animals in the first year of their life, with severe outbreaks generally Correspondence: Delia Lacasta associated with intensive sheep husbandry or transport. Orf virus belongs to the Facultad de Veterinaria de Zaragoza, C/Miguel Servet 177, Zaragoza, 50013, genus Parapoxvirus, family Poxviridae, sub-family Chordopoxvirinae.1 It is Spain a pathogen with worldwide distribution, causing significant financial losses in Tel +34 609676727 Email dlacasta@gmail.com livestock production. Transmission of orf is from direct or indirect contact with Veterinary Medicine: Research and Reports 2021:12 149–158 149 Received: 12 February 2021 © 2021 Lacasta et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. Accepted: 12 April 2021 php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For Published: 9 June 2021 permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Powered by TCPDF (www.tcpdf.org) Lacasta et al Dovepress the orf virus from pustules of infected animals containing- (cetrimide) in a gel formulation that

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red by TCPDF (www.tcpdf.org) Lacasta et al Dovepress the orf virus from pustules of infected animals containing- (cetrimide) in a gel formulation that creates a barrier virus or live vaccines, causing painful lesions in sheep and effect, numbing the pain of lesions, rapidly reducing goats, and potentially people, especially farmers and veter­ their infectivity, and hastening healing, potentially redu­ inarians. Orf is very resistant in the environment, particu­ cing the weight loss in affected individuals. The TS pro­ larly in dry atmospheres, with the virus shown to be duct was developed for pain relief in sheep undergoing infective for up to 17 years.2 Clinical presentation in Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 surgical husbandry procedures in Australia. It is also regis­ lambs or kids includes the formation of vesicles, papules, tered for use in cattle in Australia and New Zealand for pustules with a yellowish creamy and/or proliferative husbandry procedures and more recently in Laos as appearance, then scabs that finally become dry and shed, a therapy for reducing suffering and enhancing recovery usually with no scar remaining.1 Although the most com­ of lesions FMD.10–12 Registration of TS in Europe is mon lesions can be found in the oral cavity, these can be pending, although it has been shown to be efficacious in extended to the skin of the face, ears, mammary gland, the provision of pain relief and more rapid healing of feet, flanks, scrotum and perianal area.3 husbandry lesions in sheep in Australia and Spain.10,13,14 Disease diagnosis is usually based on clinical presenta­ As TS has a pH of 2.7, it has a potential viricidal impact tion. However, laboratory testing and

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ease diagnosis is usually based on clinical presenta­ As TS has a pH of 2.7, it has a potential viricidal impact tion. However, laboratory testing and confirmation may be that may reduce transmission risks, with the formulation critical as some of the differential diagnoses are notifiable potentially aiding healing and avoiding the need for other diseases, especially foot-and-mouth disease (FMD), blue­ treatments, including antibiotics.11,12 This paper describes tongue (BT), sheep pox and peste des petits ruminants a preliminary investigation of orf therapy with TS, exam­ For personal use only. (PPR). Laboratory diagnosis can be achieved through elec­ ining the potential antiviral roles and healing properties of tron microscopy, PCR based on the amplification of B2L4 the pain-relief formulation in naturally infected lambs with or VIR5 genomic regions, and/or, isolation of the virus in orf, through viral genome real-time PCR quantification cell culture using a variety of primary and continuous cell and tissue culture in ovine primary cells. lines. Vaccination is the preferred option to control the dis­ Materials and Methods ease. Live virus and attenuated vaccines usually provide All procedures were conducted under Project Licence PI adequate protection against orf virus, albeit not life-long, 40/19 approved by the Ethics Committee for Animal with their annual administration considered the best strat­ Experiments from the University of Zaragoza. The care egy to control the disease in flocks and herds.6 However, and use of animals were performed according to the orf vaccine is currently unavailable in most European Spanish Policy for Animal Protection RD53/2013, meeting countries, and where available, existing live-attenuated or the European Union Directive 2010/63

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anish Policy for Animal Protection RD53/2013, meeting countries, and where available, existing live-attenuated or the European Union Directive 2010/63 on the protection of scab-based vaccines may revert to virulence and are not animals used for experimental and other scientific fully protective. This could be due to genetic deletions purposes. present in attenuated vaccines, that may significantly reduce their immunogenicity and efficacy, and genetic Animals heterogeneity of field strains.7–9 One-month-old lambs (n=14) with similar weights and The treatment of this disorder involves standard naturally infected with orf were recruited from a farm hygiene practices and in numerous cases, management of where a natural outbreak of orf disease was occurring. presumptive secondary infections, usually with antibiotic The 1800 Lacaune sheep farm was located in a small treatments.2 As reducing antibiotic use in livestock has village of the Pyrenees mountains. Due to high prolificacy become a priority for the management of antimicrobial (2.2), some lambs were routinely artificially fed milk by an resistance (AMR) risk, there is a need for treatment pro­ artificial lactation machine. These lambs were kept in tocols that provide more effective topical treatment of orf a separate pen with proper cleaning and disinfection con­ cases. Recent studies have confirmed the efficacy of the ditions, although the farmers reported the lambs frequently wound therapy formulation Tri-Solfen® (TS) (Animal had recurrent orf infections. The lambs selected for the Ethics Pty Ltd, Australia) for reducing pain and hastening experiment were in the early stages of orf infection when healing of skin and mucosal lesions in sheep and cattle. lesions were initially diagnosed. The lambs were co- The

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e early stages of orf infection when healing of skin and mucosal lesions in sheep and cattle. lesions were initially diagnosed. The lambs were co- The dressing formulation contains two local anaesthetics located in a separate pen and randomly divided into two (lignocaine and bupivacaine), adrenaline and an antiseptic cohorts, with Group A (n=11) consisting of animals with 150 https://doi.org/10.2147/VMRR.S306355 Veterinary Medicine: Research and Reports 2021:12 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress Lacasta et al orf lesions treated with TS and Group B (n=3), an orf- haemoglobin; pg), MCHC (mean corpuscular haemoglobin infected control group remaining untreated. All lambs concentration; g/dL) and reticulocytes (K/mL). White were subjected to the same management, with artificial blood cells were assessed by counts of neutrophils (K/ feeding conducted until weaning at 45 days of age. From mL-1), lymphocytes (K/mL), monocytes (K/mL), baso­ trial commencement, the lambs also had ad libitum access phils (K/mL) and eosinophils (K/mL). Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 to compound feedstuff and straw. No other treatments were administered throughout the duration of the study. Real-Time PCR Sterile swab samples taken from the lesions at T0, before Sampling treatment, and T1, T2 and T3 were submitted to direct Sterile cotton swabs were inserted into the lesions prior to DNA extraction and further real-time PCR quantification. the application of treatments (T0) to confirm the presence Nucleic acid extraction was performed using the commer­ of orf virus. This was followed by a similar sampling of cial MagMAX™ Pathogen RNA/DNA kit (Thermo Fisher the lesions on days 1 (T1), 3 (T2)

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e commer­ of orf virus. This was followed by a similar sampling of cial MagMAX™ Pathogen RNA/DNA kit (Thermo Fisher the lesions on days 1 (T1), 3 (T2) and 5 (T3) post- Scientific) and the automated magnetic particle processor treatment of all the lesions observed on the lambs. KingFisher Flex System (Thermo Fisher Scientific), fol­ Samples of 3mL of whole blood were also collected lowing the manufacturer’s instructions. Extracted DNA from the jugular vein through a vacutainer system into samples were stored at −80°C until end of sampling in EDTA tubes to perform haematology in all the animals order to evaluate all of them in a single run of real-time prior to (T0) and ten days (T4) following treatment, PCR quantification. Orf virus detection was performed For personal use only. respectively. using the commercial qPCR kit EXOone Contagious Ecthyma (Exopol, Spain) which targets the BL2 gene Treatment Application that encodes a major envelope viral antigen. The kit con­ Following confirmation of the presence of orf virus in the tains a quantified synthetic positive control, and also an lesions, a single spray of 1.5mL of TS was applied liber­ endogenous control to avoid false-negative results. ally with a spray gun to all orf lesions in Group A lambs, Amplification was performed on a FAST 7500 cycler with the Group B lambs remaining untreated. (Applied Biosystems), and a cut-off value for positive samples was established at cycle quantification (Cq) Clinical Progression values lower than 38. Clinical examination of all lambs was performed by obser­ vers blinded to treatment. This occurred daily for 11 days Virus Culture to determine the clinical progression of the lesions, with Sterile swabs collected at T0, T1, T2 and T3 were submitted data collected on the

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s Culture to determine the clinical progression of the lesions, with Sterile swabs collected at T0, T1, T2 and T3 were submitted data collected on the changes occurring in the lesions. In to incubation with primary tissue cultures from ovine skin addition, photographic records of the lesions were taken at fibroblasts (OSF) and T-immortalized goat embryonic fibro­ three angles, including the two lateral profiles and an blasts (TIGEF). Briefly, swabs were immersed in 2mL of anterior (frontal) image with the mouth open to observe tissue culture medium, DMEM supplemented with 1% glu­ and record lesions occurring inside the mouth. Images tamine, 2% foetal bovine serum and 2% antibiotics (Sigma were also taken with a ruler to provide size comparisons. Aldrich) and then added to cells. Cells were incubated at 37° The number of lesions and the morphological appearance C, 5% CO2 atmosphere for 5 days. DNA extraction was of these lesions were chronologically recorded from each performed in cells using E.Z.N.A Blood DNA Mini Kit animal, described as papules, pustules, proliferations, (Omega bio-tek). Orf virus detection and viral load quanti­ crusts, or erosions. fication were conducted by real-time quantitative PCR in an Agilent AriaMx machine using commercial PCR EXOone Haematology Contagious Ecthyma (Exopol, Spain). Haematology was performed using an automatic haemato­ logical counter IDEXX ProcyteDx (IDEXX laboratories, Statistical Analysis of Results Westbrook, ME, USA). Measured parameters were leuco­ Statistical analyses were performed using IBM SPSS statis­ cytes (K/mL), erythrocytes (M/mL), haemoglobin (g/dL), tics version 26 (2019) software (IBM, Armonk, NY, USA). haematocrit (%), platelets (K/mL), MCV (Mean Shapiro–Wilk test was applied to quantitative

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L), tics version 26 (2019) software (IBM, Armonk, NY, USA). haematocrit (%), platelets (K/mL), MCV (Mean Shapiro–Wilk test was applied to quantitative variables for Corpuscular Volume; fL), MCH (mean corpuscular assessing normal distribution. Where normal distribution Veterinary Medicine: Research and Reports 2021:12 https://doi.org/10.2147/VMRR.S306355 151 DovePress Powered by TCPDF (www.tcpdf.org) Lacasta et al Dovepress was assessed, two ways mixed ANOVA were applied when Clinical Progression considering repeated measures (within-subject factor: Lesion progression was daily analysed by clinical examina­ repeated measure; between-subject factor: group). Where tion for eleven days following treatment in addition to quantitative variables were not normally distributed, non- a review of lesion progression from images using three parametric tests were applied (Mann–Whitney´s U-test for different angles. Firstly, the progression of the lesion surface Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 independent samples and Wilcoxon test for paired samples). was calculated with the formula of the area of the circle (πr2), Viral load data were analysed by T-Student’s test for unre­ adding the left, right and anterior surfaces of each type of lated samples. Percentages were compared by Pearson chi- lesions (papules, pustules, proliferations, crusts and erosions) square test and Fisher´s exact test. A difference was consid­ (Table 1). No differences were found in any type of lesions ered statistically significant when p≤ 0.050. throughout the study between treated and untreated lambs. In addition, the total number of each type of lesion per Results lamb was analysed, with no differences noted

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udy between treated and untreated lambs. In addition, the total number of each type of lesion per Results lamb was analysed, with no differences noted between the At sampling performed before treatment (T0), all lambs groups (Table 2). However, as expected, the total number tested positive to orf virus on real-time PCR, with Cq of papules (initial orf lesion) decreased throughout the values ranging from 23.1 to 35.9 (threshold value <38). study, whereas there was no difference in the number of Table 1 Mean and Standard Deviation (SD) for the Total Surface of Lesions (cm2), per Day, Group and Type of Lesion. Group For personal use only. A (n=11) Consisting of Animals with Orf Lesions Treated with TS and Group B (n=3), an Orf-Infected Control Group Remaining Untreated Day Group Type of Lesion Papules Pustules Proliferations Crusts Erosions 1 A 0.05±0.078 0.10±0.151 0.18±0.389 0.26±0.561 0.24±0.553 B 0.03±0.054 0.02±0.028 – 0.09±0.100 0.01±0.018 2 A 0.07±0.145 0.19±0.350 0.40±0.782 0.43±0.890 0.33±0.611 B 0.03±0.025 0.01±0.018 – 0.10±0.098 – 3 A 0.04±0.140 0.20±0.346 0.39±0.506 0.57±0.864 0.35±0.656 B – – 0.05±0.092 0.08±0.105 – 4 A 0.04±0.140 0.12±0.263 0.41±0.671 0.77±1.129 0.43±0.394 B – – 0.15±0.255 0.72±0.696 – 5 A – 0.14±0.237 0.43±0.589 0.76±1.249 0.38±0.619 B – – 0.06±0.113 0.58±1.000 – 6 A 0.08±0.248 0.08±0.176 0.33±0.541 1.19±1.118 0.65±0.999 B – – 0.36±0.464 0.58±1.000 – 7 A – 0.08±0.150 0.17±0.308 1.06±1.521 0.43±0.683 B – – 0.06±0.113 0.85±1.481 – 8 A – 0.04±0.083 0.10±0.194 1.10±1.507 0.48±0.581 B – – 0.14±0.218 1.10±1.906 0.26±0.453 9 A – 0.07±0.145 0.24±0.381 0.98±1.437 0.43±0.664 B – – – 0.58±1.000 0.26±0.453 10 A – 0.05±0.139

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07 0.48±0.581 B – – 0.14±0.218 1.10±1.906 0.26±0.453 9 A – 0.07±0.145 0.24±0.381 0.98±1.437 0.43±0.664 B – – – 0.58±1.000 0.26±0.453 10 A – 0.05±0.139 0.11±0.259 1.48±1.454 0.41±0.569 B – – – 0.41±0.708 0.26±0.453 11 A – 0.05±0.139 0.30±0.647 1.40±1.492 0.41±0.569 B – – – 0.41±0.708 0.26±0.453 152 https://doi.org/10.2147/VMRR.S306355 Veterinary Medicine: Research and Reports 2021:12 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress Lacasta et al Table 2 Mean and Standard Deviation (SD) for the Total Number of Lesions per Day, Group and Type of Lesion. Group A (n=11) Consisting of Animals with Orf Lesions Treated with TS and Group B (n=3), an Orf-Infected Control Group Remaining Untreated Day Group Number of Lesions Papules Pustules Proliferations Crusts Erosions 1 A 0.50±0.527 1.10±1.449 1.20±2.098 0.90±1.912 0.30±0.675 Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 B 1.00±1.732 0.33±0.577 – 1.00±1.00 0.33±0.577 2 A 0.30±0.483 1.00±1.886 1.40±2.066 0.90±1.370 0.60±1.075 B 0.67±0.577 0.33±0.577 – 1.33±1.528 – 3 A 0.10±0.316 0.80±1.398 1.40±1.430 0.90±1.287 0.80±1.317 B – – 1.33±2.309 0.67±0.577 – 4 A 0.10±0.316 0.40±0.699 1.00±1.491 1.30±1.636 1.40±1.776 B – – 0.33±0.577 1.67±2.082 – 5 A – 0.80±1.619 1.60±1.647 1.30±1.636 1.00±1.886 B – – 0.33±0.577 0.67±1.155 – 6 A 0.10±0.316 0.30±0.675 1.00±1.247 1.50±1.354 1.20±1.874 B – – 1.00±1.00 0.67±1.155 – For personal use only. 7 A – 0.30±0.483 0.90±1.524 1.80±2.440 1.20±2.150 B – – 0.33±0.577 1.33±2.309 – 8 A – 0.20±0.422 0.90±1.524 2.20±2.530 1.10±1.524 B – – 1.00±1.00 2.33±4.041 0.33±0.577 9 A –

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.524 1.80±2.440 1.20±2.150 B – – 0.33±0.577 1.33±2.309 – 8 A – 0.20±0.422 0.90±1.524 2.20±2.530 1.10±1.524 B – – 1.00±1.00 2.33±4.041 0.33±0.577 9 A – 0.30±0.483 1.30±1.636 2.60±4.248 0.80±1.135 B – – – 0.67±1.155 0.33±0.577 10 A – 0.20±0.422 0.70±1.059 3.70±5.143 0.50±0.707 B – – – 0.67±1.155 0.33±0.577 11 A – 0.20±0.422 0.80±1.135 3.10±4.725 0.50±0.707 B – – – 0.67±1.155 0.33±0.577 pustules and proliferations, with the number of crusts observed in erythrocytes, total leukocytes, neutrophils and increasing as days progressed. Finally, there were no dif­ monocytes on day 11 in comparison with day 1, the MCV ferences in the number of lambs presenting each type of and MCH had significantly decreased on the 11th day. lesion at each time point of the study. The number and percentage of lambs with lesion Real-Time PCR per day, group and type of lesion are displayed (Table 3). All animals tested positive in all samples, except one lamb No differences were found in any type of lesions through­ that tested negative in T2 but returned positive in T3. For out the study between treated and untreated lambs. the statistical study, the Cq values obtained at T0 and T3 were analysed, and no significant differences were Haematology observed between treated and untreated animals All blood parameters analysed remained within normal (p=0.722). The mean Cq was 29.63 in T0, 28.52 in T1, ranges throughout the entire study, with the means and 29.62 in T2 and 29.17 in T3 (Table 5). standard deviation of the haematological parameters ana­ lysed displayed (Table 4). A slight neutrophilia was Virus Culture observed on the eleventh day of study from the lambs of Cotton swabs submitted to incubation with primary tissue group A, and

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philia was Virus Culture observed on the eleventh day of study from the lambs of Cotton swabs submitted to incubation with primary tissue group A, and no significant differences were found between cultures from ovine skin fibroblasts (OSF) and groups (p>0.050). Although a significant increase was T-immortalized goat embryonic fibroblasts (TIGEF) Veterinary Medicine: Research and Reports 2021:12 https://doi.org/10.2147/VMRR.S306355 153 DovePress Powered by TCPDF (www.tcpdf.org) Lacasta et al Dovepress Table 3 Number and Percentage of Lambs That Presented the Different Type of Orf Lesions Each Day. Group A (n=11) Consisting of Animals with Orf Lesions Treated with TS and Group B (n=3), an Orf-Infected Control Group Remaining Untreated Day Group Lesion Papules Pustules Proliferations Crusts Erosions 1 A 5/10 (50%) 5/10 (50%) 4/10 (40%) 3/10 (30%) 2/10 (20%) Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 B 1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 2/3 (66.7%) 1/3 (33.3%) 2 A 3/10 (30%) 4/10 (40%) 5/10 (50%) 4/10 (40%) 3/10 (30%) B 2/3 (66.7%) 1/3 (33.3%) 0/3 (0%) 2/3 (66.7%) 0/3 (0%) 3 A 1/10 (10%) 3/10 (30%) 6/10 (60%) 4/10 (40%) 4/10 (40%) B 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 2/3 (66.7%) 0/3 (0%) 4 A 1/10 (10%) 3/10 (30%) 4/10 (40%) 6/10 (60%) 7/10 (70%) B 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 2/3 (66.7%) 0/3 (0%) A 0/10 (0%) 3/10 (30%) 6/10 (60%) 6/10 (60%) 4/10 (40%) 5 B 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 6 A 1/10 (10%) 2/10 (20%) 5/10 (50%) 7/10 (70%) 4/10 (40%) B 0/3 (0%) 0/3 (0%) 2/3 (66.7%) 1/3 (33.3%) 0/3 (0%) For personal use only. 7 A 0/10 (0%) 3/10 (30%) 3/10 (30%) 5/10 (50%) 5/10 (50%) B 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 8 A 0/10 (0%) 2/10 (20%) 3/10 (30%) 7/10 (70%) 6/10 (60%)

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(30%) 3/10 (30%) 5/10 (50%) 5/10 (50%) B 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 8 A 0/10 (0%) 2/10 (20%) 3/10 (30%) 7/10 (70%) 6/10 (60%) B 0/3 (0%) 0/3 (0%) 2/3 (66.7%) 1/3 (33.3%) 0/3 (0%) 9 A 0/10 (0%) 3/10 (30%) 5/10 (50%) 6/10 (60%) 4/10 (40%) B 0/3 (0%) 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) 10 A 0/10 (0%) 2/10 (20%) 4/10 (40%) 7/10 (70%) 4/10 (40%) B 0/3 (0%) 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) 11 A 0/10 (0%) 2/10 (20%) 4/10 (40%) 7/10 (70%) 4/10 (40%) B 0/3 (0%) 0/3 (0%) 0/3 (0%) 1/3 (33.3%) 1/3 (33.3%) showed a viral load reduction in T0 and T3 between buffalo in Laos and cattle in Cameroon.11,12 In these treated and untreated animals in both groups throughout studies, clinical observations and lesion measurements the experiment (Figures 1 and 2). However, statistical confirmed that improved welfare and healing occurred differences were only found in the viral load difference following treatment of FMD lesions, with treated cattle obtained in the case of OSF cells (p<0.05). TIGEF showed and buffalo achieving both superior appetite and lesion no difference in control animals comparing T0 and T3 healing scores with a more rapid reduction in dimensions despite treated animals displaying lower viral loads at the of lesions than other groups.11,12 In Laos, it was noted that end of the experiment (p<0.05). as the presentation of affected animals for treatment and reporting of outbreaks improved, the use of TS had poten­ Discussion tial to improve disease surveillance, reduce the socioeco­ In recent years, the novel topical anaesthetic wound for­ nomics impacts of outbreaks, and reduce risks of AMR.12 mulation TS developed for reduced pain and enhanced These studies also speculated that TS might have wound healing in surgical husbandry

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sks of AMR.12 mulation TS developed for reduced pain and enhanced These studies also speculated that TS might have wound healing in surgical husbandry procedures in live­ a viricidal effect against FMD virus if applied prior to or stock, has been suggested as potentially appropriate for the at the time of lesion rupture, potentially limiting virus management of other disorders in a variety of species.10, transmission during FMD outbreaks.12 As TS has a pH Most recently, TS was found to be efficacious for the of 2.7–2.9, if applied early in the course of the disease, it treatment of foot-and-mouth disease (FMD) in cattle and was suggested that antiviral activity might potentially limit 154 https://doi.org/10.2147/VMRR.S306355 Veterinary Medicine: Research and Reports 2021:12 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress Lacasta et al Table 4 Means and Standard Deviation (SD) of the Haematological Parameters. Group A (n=11) Consisting of Animals with Orf Lesions Treated with TS and Group B (n=3), an Orf-Infected Control Group Remaining Untreated Variable Group Day 1 Day 11 N Mean ± SD N Mean ± SD Threshold Values a b Erythrocyte count (RBC, M/µL) A 9 9.71±1.277 11 11.10±1.755 9.49–15.12 M/μL Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 B 3 9.36±9.756a 3 11.13±1.277b Haematocrit (HCT, %) A 9 31.38 ±6.824 11 30.65±6.913 27.0–42.0% B 3 29.67±8.801 3 31.00±6.243 Haemoglobin (Hb, g/dL) A 9 10.28±1.814 11 10.66±1.633 10.0–14.9 g/dL B 3 9.93±1.553 3 10.57±1.106 Mean corpuscular volume (MCV, fL) A 9 32.42±5.904a 11 27.74±5.413b 24.4–32.5 fL B 3 31.50±8.150a 3 28.03±6.550b Mean corpuscular haemoglobin (MCH, pg) A 9 10.62±1.391a 11 9.664±1.097b 8.5–11.8 pg B 3

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a 11 27.74±5.413b 24.4–32.5 fL B 3 31.50±8.150a 3 28.03±6.550b Mean corpuscular haemoglobin (MCH, pg) A 9 10.62±1.391a 11 9.664±1.097b 8.5–11.8 pg B 3 10.60±1.114a 3 9.533±1.168b Mean corpuscular haemoglobin concentration (MCHC, g/dL) A 9 22.04±2.559 11 35.28±2.734 32.3–42.0 g/dL B 3 34.50±5.112 3 34.63±4.332 For personal use only. Reticulocytes (K/µL) A 9 4.13±4.432 11 2.45±2.987 0.0–15.0 K/μL B 3 1.87±0.153 3 0.77±0.666 Leucocytes (WBC, K/µL) A 7 6.55±1.993a 10 11.06±7.86b 5.06–14.12 K/μL B 3 7.93±3.453a 3 9.17±4.788b Neutrophils (K/µL) A 7 3.66±1.575a 10 7.15±4.403b 1.17–6.11 K/μL B 3 4.83±2.695a 3 5.81±3.999b Lymphocytes (K/µL) A 7 2.73±0.960 10 3.39±1.037 2.54–9.6 K/μL B 3 2.51±0.933 3 2.41±0.925 Monocytes (K/µL) A 7 0.06±0.055a 10 0.32±0.289b 0.1–1.01 K/μL B 3 0.27±0.138a 3 0.90±0.638b Eosinophils (K/µL) A 7 0.01±0.007 10 0.08±0.111B 0.05–0.95 K/μL B 3 0.02±0.011 3 0.01±0.010A Basophil (K/µL) A 7 0.09±0.048 10 0.11±0.081 0–0.12 K/μL B 3 0.03±0.026 3 0.04±0.030 Platelets (K/µL) A 9 531.89±125.926 10 456.60±203.644 301–922 K/μL B 3 758.33±359.433 3 453.67±279.001 Notes: a,bDifferent letters mean significant differences between results in day one and day 11 (p<0.05); A,BDifferent letters mean significant differences between the two groups A and B (p<0.005). virus transmission during outbreaks. Similarly, hypocrellin shown to provide rapid and prolonged wound anaesthesia A antiviral activity seems to be related to a decline in from blockage of nociception,16 with the acidity poten­ pH.15 Low pH has been related to viricidal activity in tially sufficient to destroy virus without causing pain to the multiple models, including Herpes Simplex-1 (HSV-1) in animal. It has also been postulated

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lly sufficient to destroy virus without causing pain to the multiple models, including Herpes Simplex-1 (HSV-1) in animal. It has also been postulated that the concentration dental alginates.16 Whilst the application of acidic solu­ of lidocaine in TS is likely to be directly viricidal against tions to open wounds and ulcers is generally contraindi­ FMD virus, as at concentrations ranging from 0.5 mg/mL cated, as the acidity may exacerbate pain, the relatively (0.05%) to 100 mg/mL (10%) lidocaine blocks high concentration of lidocaine (5%) applied with bupiva­ nociception10 and has been shown to exhibit antiviral caine, adrenaline, and cetrimide in a gel matrix has been activity against the herpes virus in cell-culture and animal- Veterinary Medicine: Research and Reports 2021:12 https://doi.org/10.2147/VMRR.S306355 155 DovePress Powered by TCPDF (www.tcpdf.org) Lacasta et al Dovepress Table 5 Real-Time PCR Cq Values of Each Analysed Lamb in T0, Immediately Before Treatment, and on Days 1 (T1), 3 (T2) and 5 (T3) Post-Treatment Lamb Num. T0 T1 T2 T3 1002 28.4 30.4 33.0 30.8 Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 1003 23.1 24.6 22.6 24.0 1004 29.5 27.6 31.5 32.0 1005 24.5 25.5 25.8 25.1 1007 30.4 25.9 28.4 25.9 1008 30.3 32.1 33.4 31.4 1009 34.9 35.6 Neg. 33.0 1010 31.3 32.1 31.9 31.4 1011 25.0 23.6 27.9 30.4 Figure 2 Orf viral load in T-immortalized goat fibroblasts (TIGEF) incubated with samples from naturally infected lambs obtained from control and treated groups. 1012 35.0 29.2 30.1 26.5 Data shown are the differences in viral load from T0 to T3 in control and treated 1013 29.3 27.4 27.6 33.1 animals (p> 0.05). 1015 27.0 25.7 27.4 22.9 1016 35.9 32.0 35.5 32.3 after incubation with

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om T0 to T3 in control and treated 1013 29.3 27.4 27.6 33.1 animals (p> 0.05). 1015 27.0 25.7 27.4 22.9 1016 35.9 32.0 35.5 32.3 after incubation with OSF in tissue culture. In spite of the 1017 30.2 27.6 29.9 29.5 fact that the viral load represented in the PCR Cq values Note: Bold represents the three animals belonging to group B of untreated orf infected animals. was maintained in the five days that the study lasted, the For personal use only. reduction in the viral load shown in the culture probably model systems.17 This encouraged the present study, demonstrates that the load detected in the PCR is of already inactivated viruses. examining the impact of TS on orf lesions, another viral A reduction in healing scores was not observed in the disease that causes oral lesions. trial. However, this was expected as orf infections typi­ This study reports the first field treatment trial of orf cally trigger lesions involving abnormal ballooning of lesions using TS as a therapy for the clinical management basal epithelial cells deep in the epidermis with the rapid of contagious ecthyma. Despite necessary limitations on escalation of lesions dimensions and it was not expected numbers of animals recruited for the trial due to low that these rapidly advancing lesions could readily be availability of resources, the preliminary results provided resolved.18 However, it was postulated that the presence an encouraging indication of the potential therapeutic use of TS absorbed into the surface epithelium of lesions, of TS for reduction of infective viral load, observed as especially in early stages of the disease, could result in a significant reduction in the viral quantities detected in negative impacts on virus particles as they were released the Group A treated animals at five

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ignificant reduction in the viral quantities detected in negative impacts on virus particles as they were released the Group A treated animals at five days’ post-treatment from damaged epithelial cells, resulting in the reduced infective viral load observed. The findings from this study support the hypothesis that the early applications of TS to orf infected animals could reduce virus transmis­ sion on the farm. Potentially, this may reduce the likely appearance of new cases, the extent of animal suffering, the economic losses and the risk of zoonotic transmission of orf. However, further studies are indicated to explore this important preliminary finding. Contagious ecthyma is a highly contagious disease, and when severe outbreaks occur, up to 100% of lambs may show some degree of lesions attributable to orf infection.2 Use of TS treatment for orf potentially could control the spread of the virus, reducing the enormity of outbreaks. Figure 1 Orf viral load in ovine skin fibroblasts (OSF) incubated with swab samples Further, as the product contains an antiseptic, use could from naturally infected lambs obtained from control and treated groups. Data also diminish the risks of secondary infections, with TS shown are the differences in viral load from T0 to T3 in control and treated animals (*p< 0.05). offering a non-antimicrobial therapeutic option for treating 156 https://doi.org/10.2147/VMRR.S306355 Veterinary Medicine: Research and Reports 2021:12 DovePress Powered by TCPDF (www.tcpdf.org) Dovepress Lacasta et al clinical orf lesions, potentially reducing the risk of AMR cultures from ovine skin fibroblasts and TIGEF, tissue that occurs with antimicrobial use in current orf therapy. cultures from T-immortalized goat embryonic fibroblasts. Of interest, was a recent

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, tissue that occurs with antimicrobial use in current orf therapy. cultures from T-immortalized goat embryonic fibroblasts. Of interest, was a recent observation that a lower rate of secondary infection occurred following application of TS Acknowledgments during surgical tail-docking of lambs, enabling the con­ We would like to acknowledge the farmers Marcos Périz Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 sideration of replacing routine antibiotic cover with this Bestué, Antonio Périz Barbanoj and María Jesús Bestué topical anaesthetic and antiseptic wound formulation.14 Noeguero that allows us to work with the animals on their The positive response to therapy observed in two farm. reported studies on the use of this product for clinical FMD, accompanied by an absence of adverse side-effects, suggest this novel approach to viral therapy is worthy of Funding further consideration, particularly if the findings in this pre­ This research was supported by funding and product from liminary trial of viricidal activity are found to be consistent the Australian company Animal Ethics Pty Ltd. The work in future studies. The product invokes positive and pro­ was also supported by the Aragón Government and the longed pain-relieving and wound healing effects in livestock European Social Fund (A15_17R, Construyendo Aragón following blockage of nociception during treatment of 2016-20) and Project CONECTIM funded by Gobierno de wounds and lesions from aversive husbandry procedures Navarra (PC052-053). and epitheliotropic virus infections, as described.10–12,19–23 For personal use only. This novel approach to the treatment of mucosal and skin Disclosure viral lesions in ruminants can

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ns, as described.10–12,19–23 For personal use only. This novel approach to the treatment of mucosal and skin Disclosure viral lesions in ruminants can potentially lead to reductions The abstract of this paper was presented at the in the transmission of the disease, with positive improve­ International Congress on the breeding of sheep and ments in animal welfare and reduction of AMR risk. Further goats. A hybrid conference that was held in Bonn, as an studies on the role of topical anaesthetic lesion therapy in oral presentation with interim findings. The authors report managing some of the most important viral diseases of small no conflicts of interest in this work. and large ruminants are proposed. References Conclusion 1. Nandi S, De Ujjwal K, Chowdhury S. Current status of contagious ecthyma or orf disease in goat and sheep—a global perspective. This is the first scientific report of the application to orf Small Rum Res. 2011;96:73–82. doi:10.1016/j. lesions in lambs during an outbreak, of a novel pain-relief smallrumres.2010.11.018. 2. Spyrou V, Valiakos G. Orf virus infection in sheep and goats. Vet and wound-healing therapy developed for managing aver­ Microbiol. 2015;181:178–182. doi:10.1016/j.vetmic.2015.08.010. sive livestock procedures. Although no significant differ­ 3. Reid HW. Orf. In: Martin WB, Aitken ID, editors. Diseases of Sheep. ences were found in the reduction of lesion scores, the 3rd ed. Oxford: Blackwell Science; 2000. 4. Inoshima Y, Murakami K, Yokoyama T, et al. Genetic heterogeneity significant decrease of infective viral load in the treated among parapoxviruses isolated from sheep, cattle and Japanese serows group is encouraging, suggesting further studies on the use (Capricornis crispus). J Gen Virol. 2000;82:1215–1220.

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m sheep, cattle and Japanese serows group is encouraging, suggesting further studies on the use (Capricornis crispus). J Gen Virol. 2000;82:1215–1220. doi:10.1099/ 0022-1317-82-5-1215. of TS for treatment of orf lesions in small ruminants are 5. Kottaridi C, Nomikou K, Lelli R, et al. Laboratory diagnosis of warranted. This innovation could alter the paradigm of orf contagious ecthyma: comparison of different PCR protocols with treatments that have traditionally focused on using inap­ virus isolation in cell culture. J Virol Methods. 2006;134:119–124. doi:10.1016/j.jviromet.2005.12.005. propriate and expensive antimicrobials, to one of improv­ 6. Lacasta D, Ferrer LM, Ramos JJ, et al. Vaccination schedules in small ing the welfare of animals suffering from a viral disease. ruminant. Vet Microbiol. 2015;181:34–46. doi:10.1016/j. vetmic.2015.07.018. This innovation may provide both clinical benefits to 7. Musser J, Taylor CA, Guo J, et al. Development of a contagious affected animals, reduce AMR and food-safety risks, plus ecthyma vaccine for goats. Am J Vet Res. 2008;69:1366–1370. potentially, reduce viral transmission loads and the sever­ doi:10.2460/ajvr.69.10.1366. 8. Musser JMB, Waldron DF, Taylor CA. Evaluation of homologous and ity of outbreaks. heterologous protection induced by a virulent field strain of Orf virus and an Orf vaccine in goats. Am J Vet Res. 2012;73:86–90. doi:10.2460/ajvr.73.1.86. Abbreviations 9. da Costa RA, Cargnelutti JF, Schild CO, et al. Outbreak of contagious ecthyma caused by Orf virus (Parapoxvirus ovis) in a vaccinated sheep FMD, foot-and-mouth disease; TS, pain-relief product flock in Uruguay. Braz J Microbiol. 2019;50:565–569. doi:10.1007/ (Tri-Solfen); AMR, antimicrobial resistance; OSF, tissue s42770-019-00057-7.711

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duct flock in Uruguay. Braz J Microbiol. 2019;50:565–569. doi:10.1007/ (Tri-Solfen); AMR, antimicrobial resistance; OSF, tissue s42770-019-00057-7.711 Veterinary Medicine: Research and Reports 2021:12 https://doi.org/10.2147/VMRR.S306355 157 DovePress Powered by TCPDF (www.tcpdf.org) Lacasta et al Dovepress 10. Roberts CD, Windsor PA. Innovative pain management solutions in 17. Haines HG, Dickens CB, Brigham DP. Antiviral pharmaceutical animals may provide improved wound pain reduction during debri­ preparations and methods for their use. Patent US 4628063A. 1986. dement in humans: an opinion informed by veterinary literature. 18. Windsor PA, Nampanya S, Tagger A, et al. Is Orf virus a risk to Int Wound J. 2019;16(4):968–973. doi:10.1111/iwj.13129. expanding goat production systems in developing countries? A study 11. Lendzele S, Mavoungou J, Burinyuy K, et al. Efficacy and applica­ from Lao PDR. Small Rum Res. 2017;154:123–128. doi:10.1016/j. tion of a novel topical anaesthetic wound formulation for treating smallrumres.2017.08.003. cattle with foot-and-mouth disease: a field trial in Cameroon. 19. Lomax S, Sheil M, Windsor PA. Impact of topical anaesthesia on Veterinary Medicine: Research and Reports downloaded from https://www.dovepress.com/ by 123.255.209.218 on 09-Jun-2021 Transbound Emerg Dis. 2020;00:1–12. doi:10.1111/tbed.13923. pain alleviation and wound healing in lambs after mulesing. Aus Vet 12. Windsor PA, Earp F, Mac Phillamy I, et al. Managing welfare and J. 2008;86:159–168. doi:10.1111/j.1751-0813.2008.00285.x. antimicrobial-resistance issues in treating foot-and-mouth disease 20. Paull DR, Lee C, Colditz IG, et al. The effect of a topical anaesthetic lesions: a new therapeutic approach. Vet Med Res Rep. formulation, systemic flunixin and

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Lee C, Colditz IG, et al. The effect of a topical anaesthetic lesions: a new therapeutic approach. Vet Med Res Rep. formulation, systemic flunixin and carprofen, singly or in combina­ 2020;11:99–107. doi:10.2147/VMRR.S273788. tion, on cortisol and behavioural responses of Merino lambs to 13. Windsor PA, Lomax S, White P. Progress in pain management to mulesing. Aus Vet J. 2009;85:98–106. doi:10.1111/j.1751- improve small ruminant farm welfare. Small Rum Res. 0813.2009.00429.x 2016;142:55–57. doi:10.1016/j.smallrumres.2016.03.024. 21. Lomax S, Dickson H, Sheil M, et al. Topical anaesthesia alleviates 14. Ferrer LM, Lacasta D, Ortín A, et al. Impact of a topical anaesthesia short-term pain of castration and tail docking in lambs. Aus Vet J. wound management formulation on pain, inflammation and reduction 2010;88:67–74. doi:10.1111/j.1751-0813.2009.00546.x of secondary infections after tail docking in lambs. Animals. 2020;10 22. Lomax S, Sheil M, Windsor PA. Duration of action of a topical (8):1255. doi:10.3390/ani10081255 anaesthetic formulation for pain management of mulesing in sheep. 15. Chaloupka R, Sureau F, Kocisova E, et al. Hypocrellin Aus Vet J. 2013;91:160–167. doi:10.1111/avj.12031. A photosensitization involves an intracellular pH decrease in 3T3 23. Windsor PA, Lomax S. Addressing welfare concerns in control of cells. Photochem Photobiol. 1998;68(1):44–50. doi:10.1111/j.1751- ovine cutaneous myiosis in sheep in Australia. Small Rum Res. 1097.1998.tb03251.x. 2013;110:165–169. doi:10.1016/j.smallrumres.2012.11.027. 16. Nallamuthu N, Braden M, Oxford J, et al. Modification of pH con­ ferring viricidal activity on dental alginates. Materials. 2015;8 For personal use only. (4):1966–1975. doi:10.3390/ma8041966. Veterinary Medicine: Research and

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ricidal activity on dental alginates. Materials. 2015;8 For personal use only. (4):1966–1975. doi:10.3390/ma8041966. Veterinary Medicine: Research and Reports Dovepress Publish your work in this journal Veterinary Medicine: Research and Reports is an international, peer- and includes a very quick and fair peer-review system. Visit reviewed, open access journal publishing original research, case http://www.dovepress.com/testimonials.php to read real quotes from reports, editorials, reviews and commentaries on all areas of veterinary published authors. medicine. The manuscript management system is completely online Submit your manuscript here: http://www.dovepress.com/veterinary-medicine-research-and-reports-journal 158 DovePress Veterinary Medicine: Research and Reports 2021:12 Powered by TCPDF (www.tcpdf.org)