Efficacy and application of a novel topical anaesthetic wound formulation for treating cattle with Foot-and-Mouth disease: A field trial in Cameroon

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Received: 15 September 2020 | Revised: 25 October 2020 | Accepted: 9 November 2020 DOI: 10.1111/tbed.13923 ORIGINAL ARTICLE Efficacy and application of a novel topical anaesthetic wound formulation for treating cattle with Foot-and-Mouth disease: A field trial in Cameroon Sevidzem S. Lendzele1,2 | Jacques F. Mavoungou1,3 | Kong A. Burinyuy4 | Koumba A. Armel1 | Simon J. Dickmu5 | James R. Young6 | Peter C. Thomson7 | Peter A. Windsor6 1 Institut de recherche en Ecologie Tropicale (IRET-CENAREST), Libreville, Gabon Abstract 2 Ecole Doctorale des Grandes Ecoles Recently, a wound dressing formulation, (Tri-Solfen®, Medical Ethics Pty Ltd, (EDGE), Libreville, Gabon Australia; TS) registered for use in ruminant husbandry in Australia, was registered 3 Université des Sciences et Techniques (USTM), Franceville, Gabon for Foot-and-Mouth Disease (FMD) therapy in large ruminants in Laos, following 4 School of Veterinary Science and Medicine, clinical observations of improved welfare and healing following treatment of FMD University of Ngaoundéré, Ngaoundéré, lesions. In November 2019, an FMD outbreak in Cameroon provided an opportunity Cameroon 5 for a field trial, comparing clinical responses and recoveries to treatments on a sam- The National Veterinary Laboratory (LANAVET), Garoua North, Cameroon ple of cattle (n = 36) comprising three equal groups of animals (n = 12), comparing 6 Sydney School of Veterinary Sciences, responses to three treatments: (i) the application to lesions of TS, (ii) the administra- The University of Sydney, Camden, NSW, Australia tion of parenteral oxytetraycline commonly used for FMD in Cameroon; and (iii) an 7 School of Life and Environmental Sciences, untreated control group (C). Appetite scores, lesion healing scores, and changes in The

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oon; and (iii) an 7 School of Life and Environmental Sciences, untreated control group (C). Appetite scores, lesion healing scores, and changes in The University of Sydney, Camden, NSW, dimensions of lesions, were recorded over a 15-day study period. Cattle treated with Australia TS achieved both superior appetite and lesion healing scores with more rapid reduc- Correspondence tion in dimensions of lesions than other groups. Farmer observations indicated the TS Peter A. Windsor, Sydney School of Veterinary Sciences, The University of treatment group had a more rapid return to eating with cessation of excessive saliva- Sydney, Camden, NSW, Australia. tion, and more rapid return of mobility (walking) with absence of overt lameness. The Email: peter.windsor@sydney.edu.au findings indicate that although mortality is usually low in FMD outbreaks, the disease is a debilitating and painful disorder with negative animal welfare impacts that should be addressed. All farmers expressed their desire that the product be made available for use in their region and modelling indicates that TS therapy imposes no additional financial burden on farmers, with the treatment likely to be provided at a similar or reduced cost to current treatment choices. As use of antibiotics for treatment of a viral disease potentially increases pressures for development of antimicrobial resist- ance and residues in the food chain, TS as an alternative non-antimicrobial therapy should be promoted for wider use in FMD outbreaks. KEYWORDS animal welfare, Cameroon, cattle, foot-and-mouth disease, therapeutic efficacy This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly

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ms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH Transbound Emerg Dis. 2020;00:1–12. wileyonlinelibrary.com/journal/tbed | 1 2 | LENDZELE et al. 1 | I NTRO D U C TI O N Recently, an Australian wound dressing formulation, Tri-Solfen® (Medical Ethics Pty Ltd, Australia; TS) registered for use in cattle Cameroon, with a population of almost 28 million people, is located and small ruminant husbandry in Australia and New Zealand, was in central Africa, bordering the Gulf of Guinea. It is a key transit link, registered for FMD therapy in large ruminants in Laos in south east sharing borders with six countries: Nigeria, Chad, Central African Asia (Windsor et al., 2020). The wound and lesion dressing formu- Republic, Republic of Congo and Equatorial Guinea. The gross do- lation contains two local anaesthetics (lignocaine and bupivacaine), mestic product (GDP) per capita of Cameroon is estimated at USD adrenalin and cetramide in a gel matrix that creates a barrier effect, 3,700 (CIA, 2017). The approximately 7.1 million cattle in Cameroon numbing the pain of lesions, rapidly reducing their infectivity, and are susceptible to regular outbreaks from Foot-and-Mouth Disease hastening healing, potentially reducing the weight loss in affected (FMD), and endemic disease in much of Africa. With no preventive individuals (Windsor et al., 2020). If made available for purchase control programmes in place and no access to commercial FMD vac- and administration by farmers, this product could provide a viable cination, farmers are focused on treatment choices for affected cat- alternative approach

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ministration by farmers, this product could provide a viable cination, farmers are focused on treatment choices for affected cat- alternative approach for managing FMD in Cameroon, other African tle, with antibiotics and/or traditional therapies commonly used and countries and developing countries globally. This formulation offers prolonged periods for animals to recover. numerous advantages over current therapies as it provides effi- FMD is a most important global viral pathogen of artiodactyl cacious pain relief and more rapid healing of wounds and lesions farmed and wildlife animals. The disease is characterized by le- (Roberts & Windsor, 2019; Windsor et al., 2016, 2020). Further, sions in and around the mouth and feet (Fakhrul-Islam et al., 2016). with a pH of ~2.7, and containing the antiseptic cetrimide, it po- Globally, there are seven pools of circulating FMD viruses recog- tentially has viricidal impacts and anti-bacterial properties respec- nized. Each pool represents independently circulating and evolving tively, avoiding the need for other treatments, including antibiotics FMD virus (FMDV) genotypes. Within the pools, cycles of emer- (Windsor et al., 2020). gence and spread occur that usually affect multiple countries in the In November 2019, TS was provided to the Cameroon research region. In the absence of specific and laboratory-confirmed reports, team for trials during FMD outbreaks in cattle involving the extensive it should be assumed that the prevalent serotypes are continuously smallholder cattle raising system. There are three cattle husbandry circulating in parts of the pool area and would be detected if suffi- systems in Cameroon, including: (1) intensive, with animals raised in cient surveillance was in place. Cameroon sits in the West/Central

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ted if suffi- systems in Cameroon, including: (1) intensive, with animals raised in cient surveillance was in place. Cameroon sits in the West/Central pens and supplied grass and food supplements (e.g. cotton cakes) African region designated as Pool 5. FMD causes huge economic without access to pastures and where attendance by veterinarians losses in Cameroon, with estimation of the total annual cost of FMD is common; (2) semi-intensive, with animals kept in traditional pens management at USD112 million (FAO, 2015). that also enable them to access pastures freely; and (3) extensive, Of the seven FMDV serotypes, four (O, A, SAT 1 & SAT2) occur with animals pastured freely through the day, then returned to tradi- in Cameroon (Ehizibolo et al., 2019; Lendzele, Abdoulmoumini, tional pens before dusk where they spend the night, rarely receiving et al., 2019; Ludi et al., 2016). There is no mass vaccination programme food supplements or attended by a veterinary technician (para-vet- for FMD and no commercial FMD vaccines are available in Cameroon erinarian) or veterinarian. (Bertram et al., 2018). A pilot trial using commercial trivalent vaccine This report describes a field trial conducted on extensive cattle (Aftovax®) was conducted in 2015 in Ngaoundere, with observations farms in Cameroon aimed at evaluating the efficacy of this therapy that clinical infection of FMD appeared to have been prevented, al- for FMD lesion management, particularly for enhancing recovery though subclinical persistent infection occurred lasting approximately and wound healing. The study compared the clinical responses from one month, as confirmed by field observations (Bertram et al., 2018). TS therapy to the most commonly used and available antimicrobial Cattle owners in Cameroon routinely

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med by field observations (Bertram et al., 2018). TS therapy to the most commonly used and available antimicrobial Cattle owners in Cameroon routinely manage the disease using a range therapy currently in use, parenteral oxytetracycline. If TS is proven of therapies, including antibiotics, anti-inflammatory preparations and to be as efficacious for FMD in this current study as it appeared to be traditional formulations (Lendzele, Marvoungou et al., 2019). on first use in Laos as described (Windsor et al., 2020), it is likely that In the absence of strategic preventive control programmes, there the product could offer an important innovation for improving FMD are urgent needs for alternative FMD management options for en- lesion treatment, potentially globally, with reduced animal welfare demic FMD-infected countries, including Cameroon. With afford- burdens, risks of AMR issues and possibly, increased transboundary able and efficacious vaccine candidates for managing FMD unlikely disease reporting and surveillance. in the near future, the review and rationalizing of FMD therapies are advisable. Several topical treatments with ethno-veterinary and recognized veterinary pharmaceuticals have been examined in 2 | M ATE R I A L S A N D M E TH O DS FMD endemic settings in Africa (Al-Lethie et al., 2018; Fakhrul-Islam et al., 2016; Gakuya et al., 2011; Misk et al., 2015). However, the 2.1 | Ethics statement widespread use of parenteral antibiotics for FMD globally, including numerous countries in Africa and Cameroon in particular, presents In addition to adopting current procedures on animal and human eth- antimicrobial resistance (AMR) and potential food safety residue ics processes in Cameroon, this collaboration ensured compliance risks that need to be addressed. with the

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nce (AMR) and potential food safety residue ics processes in Cameroon, this collaboration ensured compliance risks that need to be addressed. with the National Health and Medical Research Council's (NHMRC) LENDZELE et al. | 3 National Statement on Ethical Conduct in Human Research (2007) day, the outbreak areas were accessed, confirming the typical lesions and the Universities Australia Australian Code for the Responsible of vesicular to ulcerative glossitis and coronitis consistent with clini- Conduct of Research. This included ensuring that all participants cal signs of FMD. The treatment trials commenced immediately. Two provided verbal informed consent for the administration of thera- teams of two members each were formed to enable animal treatments pies, collection of animal samples, observations and interviews, plus and follow-up monitoring of the treated cases. Participating farmers participation in videos and images, where written consent was una- were selected following demonstration of owner willingness to par- vailable due to farmer illiteracy. ticipate, and included extensive smallholder cattle farms (designated as T1, T2 etc) from the villages of Horé Mayanga, Borongo, Tchabal Baouro and Mbidjoro. The GPS data of all farms were recorded. 2.2 | Trial location and design As Moore-Oxy® (MO; oxytetracycline HCL 5%, Hebei Kexing Pharmaceutical Co. Ltd., Shijiazhuang City, China) was the reference This clinical trial was conducted between the 5th of November and the parenteral antibiotic commonly used by farmers to manage clinical 5th of December 2019 in Ngaoundere II, Ngaoundere III and Martap FMD in the study area (Lendzele, Marvoungou, et al., 2019), it was subdivisions in the Vina Division of the Adamawa plateau. Around decided to compare the efficacy of

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area (Lendzele, Marvoungou, et al., 2019), it was subdivisions in the Vina Division of the Adamawa plateau. Around decided to compare the efficacy of this formulation on the healing 40% of the cattle population of Cameroon is located in the Adamawa of FMD lesions of the coronary band with that of a single topical region (MINEPIA, 2013). Ngaoundere is the capital of the Adamawa treatment with TS. As MO is administered intra-muscularly and TS region, with geo-referenced coordinates between 6° 40' 0" and 7° 30' is administered topically, on each farm, three animals were matched 0" north latitude and between 13° 20' 0" and 14° 10' 0" east longitude by age and breed, with similar FMD clinical presentations; with one (Figure 1). An FMD outbreak in the municipalities of Ngaoundere II, treated with MO, one with TS and the third left untreated. This cre- Ngaoundere III and Martap was advised to the lead author by a field ated 12 sets of cattle comprising 3 animals each for this comparative technician in Ngaoundere on the 4th of November 2019. The following clinical trial on the different farms (Table 1). F I G U R E 1 Maps showing the study sites (Ngaoundere II, Ngaoundere III and Martap). M1: Mbidjoro 1, M2: Mbidjoro 2, T1: Tchabal Baouro 1, T2: Tchabal Baouro 2, T3: Tchabal Baouro 3, T4: Tchabal 4, T5: Tchabal 5, T6: Tchabal 6, B: Borongo, H1: Horé Mayanga 1, H2: Horé Mayanga 2, H3: Horé Mayanga 3 4 | LENDZELE et al. TA B L E 1 Age and breed of cattle recruited into each treatment • number of days until cattle mobility (walking) returned; group • number of days cattle were not eating; Treatment group • number of days cattle continued to salivate in response to the presence of oral lesions; Moore- Tri- • number of days cattle displayed lameness, reflecting the

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days cattle continued to salivate in response to the presence of oral lesions; Moore- Tri- • number of days cattle displayed lameness, reflecting the likely Variable Oxy® Solfen® Control Overall progress in healing of painful foot lesions; and Average age (year) 3.7 4 3.3 3.7 • number of days required for cattle to return to grazing on pasture. Age range (year) 2 to 5 2 to 8 2 to 5 2 to 8 Breed count Goudali 9 9 10 28 2.5 | Treatment cost model Holstein 1 1 1 3 White Fulani 1 2 1 4 The costs of treatments were estimated and used to develop a cost- Red Fulani 1 0 0 1 benefit model enabling comparison of therapies that could assist de- Total 12 12 12 36 cisions for both individual farmers and public health policy makers on FMD outbreak management. 2.3 | Treatment applications 2.6 | Statistical analyses The clinical treatment trial was conducted with animals displaying ‘fresh’ oral and coronary band FMD lesions, observed as intact vesi- 2.6.1 | Ordinal scale analysis cles or recently ruptured vesicles. Of the 12 farmers enrolled into the 15-day treatment trial with three matched-cattle, each received Both the lesion healing scores and the appetite scores were recorded on a different treatment, with the age and breed of the cattle recruited a four-point ordinal scale, not a quantitative scale, so appropriate ordi- into each treatment group presented (Table 1). nal categorical methods were required for this analysis (Agresti, 2002). Animals treated with TS had all lesions liberally sprayed with a single In addition, walking was also modelled on a three-point ordinal scale: topical application of up to 2 ml of the product, as per label instructions. (1: immobile; 2: walking with difficulty; 3: walking normally). An ordi- Animals treated with MO received

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l of the product, as per label instructions. (1: immobile; 2: walking with difficulty; 3: walking normally). An ordi- Animals treated with MO received intra-muscular injections of the nal logistic mixed model was fitted to each data set with fixed effects product daily for 3 days, also at doses per label instructions (2-4 mg/kg). for Treatment, Day, Breed and Age (covariate), and a random effect for Clinical response observations were initially conducted and recorded by the individual Animal ID. A Treatment × Day interaction was included veterinary technicians with experience of observing animals with clini- in each of the models, to allow for a different shaped time course for cal FMD and these personnel trained the farmers in how to conduct the each treatment. Note that the fitted model returned a set of model- required observations. All assessors were blind to treatment to avoid based probabilities of obtaining each possible score (1 through 4, or 1 bias. The observations included the recording of any improvements in through 3), for the particular combination of terms in the model. The demeanour and interest and capacity of the animal to walk and eat, plus model was fitted using the clmm function in the ordinal package of R the following semi-quantitative clinical measurements: (Christensen, 2019), and probability estimates obtained using the em- means (Lenth, 2020) and RVAideMemoire (Hervé, 2020) packages in R. • coronary band lesion healing score (LHS) on a scale of 1 to 4 (adapted from Al-Lethie et al., 2018): • coronary band lesion size (cm): healing of coronary band lesions 2.6.2 | Binary data analysis was measured quantitatively in the field using a 30 cm ruler, every 3 days from day zero (D0) through day 15 (D15), with lesions pho- The status of cattle

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measured quantitatively in the field using a 30 cm ruler, every 3 days from day zero (D0) through day 15 (D15), with lesions pho- The status of cattle being on pasture and cattle salivating (Yes = 1, tographed using android mobile phones; and No = 0) is binary outcomes, and for this, binary logistic mixed mod- • appetite score (AS) on a scale of 1 to 4, reflecting both interest in els were used to analyse these data. As above, fixed effects for eating, noting that animals with fever generally display anorexia, and Treatment, Day, Treatment × Day, Age, Breed and Sex, and a random ability to eat, noting that animals with oral ulceration may not have effect for Animal ID were included in the model. The model was fitted the capacity to prehend (adapted from Al-Lethie et al., 2018). using the glmer function in the lme4 package of R (Bates et al., 2015), and model-based means obtained using the emmeans package. 2.4 | Farmer observations 2.6.3 | Quantitative data analysis The farmers were also blind to treatment and after training, were re- quested to provide their observations for each animal for each treat- Lesion size (cm) was analysed using a linear mixed model, with fixed ment group, with the following information recorded: effects for Treatment, Day, Treatment × Day, Age, Breed and Sex, LENDZELE et al. | 5 and a random effect for Animal ID. Model fitting was via the lmer 3.2 | Appetite scores function in the lme4, and model-based means obtained using the emmeans package. Event duration data (number of days that cat- Again, there was a highly significant Treatment × Day interaction tle are immobile, walk with difficulty, walk normally, on pasture, (p = 2.0 × 10–6) for appetite score, indicating treatment effects were and salivating) were analysed using linear

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fficulty, walk normally, on pasture, (p = 2.0 × 10–6) for appetite score, indicating treatment effects were and salivating) were analysed using linear models with fixed ef- changing over the study period. Model-based appetite score prob- fects for Treatment, Age, Breed and Sex. Due to the positive skew, abilities are displayed (Figure 3). Most control cattle had no appe- a loge(y + 1) transformation was applied. However, with the large tite with low scores over the duration of the study period (score of number of ‘zero’ durations, hypothesis testing was conducted with 1). However, the two treatment groups had moderate (MO) to high permutation tests rather than F tests, using the aovperm function in (TS) scores on Day 0, low on Day 3, then progressively increasing the permuco package (Frossardm & Renaud, 2019) in R. scores after that. After Day 0, there were no significant differences in scores between MO and TS-treated cattle (all p > .10). Neither Age (p = .810), Breed (p = .524), nor Sex (p = .324) had a significant effect 3 | R E S U LT S in appetite score. 3.1 | Lesion healing scores 3.3 | Lesion size There was a highly significant Treatment × Day interaction (p = 3.4 × 10–7), indicating differing lesion healing score time courses There was a highly significant Treatment × Day interaction (p < 2 × across the three treatment groups. Model-based lesion score prob- 10–16) for lesion size (Figure 4), indicating different rates of healing abilities are displayed (Figure 2). While control cattle maintained the across the three groups. Initially, there were no significant differ- presence of erosions/ulcers (a score of 1), recoveries were observed ences in mean wound sizes amongst the three groups (all p > .25). in the two treatment groups. From Day 9

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ers (a score of 1), recoveries were observed ences in mean wound sizes amongst the three groups (all p > .25). in the two treatment groups. From Day 9 onwards, cattle treated While mean wound size increased for Control cattle, they reduced with TS had significantly higher lesion healing scores than those on for the two treatment groups, with those in the TS treatment reduc- MO (Day 9: p = .025; Day 12: p = .016; Day 15: p = .0008). None ing at a faster rate and differences were significant from Day 9, (all of the other terms had a significant association with lesion healing p < .01). There was no significant effect of Age (p = .454), Breed score: Age: p = .464; Breed: p = .311; Sex: p = .376. (p = .225), nor Sex (p = .374) on lesion size. F I G U R E 2 Model-based probabilities of obtaining lesion healing scores 1 through 4 across the six study days, for the three treatment groups 6 | LENDZELE et al. F I G U R E 3 Model-based probabilities of obtaining appetite scores 1 through 4 across the six study days, for the three treatment groups F I G U R E 4 Model-based mean lesion size (cm) across the six study days, for the three treatment groups. Shaded areas are ±1 SE of the mean LENDZELE et al. | 7 3.4 | Walking cattle having mean duration 3.72 ± 1.14 days, compared with female duration of 1.82 days. When walking is considered as an ordinal score (Immobile < With difficulty < Normal), there was a highly significant Treatment × Day interaction (p = 1.8 × 10–13). Initially (Day 0), there were no differ- 3.5 | Time on pasture ences in walking scores between the three groups (all p > .15) and some cattle remained ‘Immobile’ or ‘With difficulty’ throughout There was a highly significant Treatment × Day interaction for the the study period (Figure 5). However,

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ed ‘Immobile’ or ‘With difficulty’ throughout There was a highly significant Treatment × Day interaction for the the study period (Figure 5). However, walking improved for the presence of cattle accessing pasture (p = 1.5 × 10–6). It was apparent two treatment groups, particularly for the TS treatment group. that Control cattle basically never returned onto pasture during the However, the differences between these two groups never reached trial (Figure 6). However, for the two treatment groups, after initially threshold significance (all p > .05). There were no significant ef- none being on pasture, all were on pasture by Day 6. Although TS fects of other factors on walking (Age: p = .583; Breed: p = .9012; showed a faster return to pasture than MO, it was not possible to Sex: p = .778). formally test this. There were no significant effects of Age (p = .791) An additional analysis was conducted to compare the number nor Sex (p = .661) on the probability of being on pasture; there was of days that cattle are immobile, number of days they walk with some evidence of breed differences (p = .023), although breed es- difficulty, and number of days they walk normally (Table 2). In all timates could not be relied upon because of small number in some cases, these durations differed significantly between the three breeds. treatment groups (all p < .01). Control cattle had a significantly lon- ger period of immobility compared with the two treatment groups (both p < .005). 3.6 | Salivation TS-treated cattle had a significantly shorter period of walking with difficulty compared with Control cattle (p = .0022), and also There was no significant Treatment × Day interaction for occurrence of had significantly longer period walking normally compared with both salivation

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There was no significant Treatment × Day interaction for occurrence of had significantly longer period walking normally compared with both salivation (p = .496), nor was there an overall main effect of Treatment Control and MO-treated cattle (both p < .005). Note that there were (p = .401). Model-based probabilities of salivating are shown (Figure 7). no effects of Age, Breed, not Sex on the three durations, with the However, from initial high rates of salivating, there was a significant exception of Sex on duration of normal walking (p = .033) with male decline over the study period (p = 6.4 × 10–12), with an apparent faster F I G U R E 5 Model-based probabilities of obtaining walking scores of ‘Immobile’, ‘With difficulty’ and ‘Normal’ across the six study days, for the three treatment groups 8 | LENDZELE et al. TA B L E 2 Analysis of the number of Walking Variable p-Value Control Moore-Oxy® Tri-Solfen® days that cattle are immobile, number of Immobile Treatment .002 2.47A± 1.39 0.05B ± 0.35 0.00 B ± 0.30 days walking with difficulty and number Age .942 of days walking normally Breed .590 Sex .268 With difficulty Treatment .009 8.27A ± 4.95 3.49AB ± 2.00 0.70 B ± 0.85 Age .471 Breed .875 Sex .724 Normal Treatment .000 0.60A ± 0.40 2.72B ± 0.78 7.18C ± 1.92 Age .221 Breed .117 Sex .033 Note: Means sharing the same superscript alphabets are not significantly different (p > .05). F I G U R E 6 Model-based probabilities of cattle being on pasture across the six study days, for the three treatment groups. Shaded areas are ±1 SE of the mean (where available) rate of decline for the two treatment groups compared with the con- 3.7 | Cost of therapy model trol group. There were no significant effects of Age (p = .619), Breed (p = .096) nor Sex (p =

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oups compared with the con- 3.7 | Cost of therapy model trol group. There were no significant effects of Age (p = .619), Breed (p = .096) nor Sex (p = 1.000) on instances of salivation. The treatment types and costs for FMD therapy in Cameroon were The results of the analysis to compare the number of days that estimated, enabling a model to support therapy decisions for both cattle were salivating are displayed (Table 3). As in the previous anal- individual farmers and public health policy makers (Table 4). With ysis, no significant effects of Treatment, nor Age, Breed or Sex were a single treatment of 1ml per lesion for TS, at USD0.50 per ml, detected (all p > .1), with the two treatment groups spending less the cost of treatment per animal is estimated between USD1.50 time salivating. and 2.50. LENDZELE et al. | 9 F I G U R E 7 Model-based probabilities of cattle salivating across the six study days, for the three treatment groups TA B L E 3 Analysis of the number of Variable p-Value Control Moore-Oxy® Tri-Solfen® days that cattle are salivating Treatment .146 1.59A ± 0.82 0.49A ± 0.39 0.56A ± 0.46 Age .171 Breed .537 Sex .500 Note: Means sharing the same superscript alphabets are not significantly different (p > .05). TA B L E 4 Treatment types used by Est. cost/day Est. cost/ farmers, application method, days of Treatment choice Application (USD) Treatment days animal (USD) treatment and estimated daily and total costs Moore-Oxy Injection 0.85 3 2.55 Procaine Injection 0.17 3 0.51 penicillin Oxytet 30% Injection 0.85 3 2.55 Survidium Injection 0.85 5 4.25 Insecticide & Topical 0.42 7 2.94 Petrol Traditional drugs Topical/oral 0.51 7 3.57 OXYDOZER 50 Injection 0.85 3 2.55 Tri-Solfen Topical / 1 2.50 ® ® Note: Moore-Oxy has a 7 day milk WHP and 21 meat WHP.

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drugs Topical/oral 0.51 7 3.57 OXYDOZER 50 Injection 0.85 3 2.55 Tri-Solfen Topical / 1 2.50 ® ® Note: Moore-Oxy has a 7 day milk WHP and 21 meat WHP. Tri-Solfen has a recommended 4 day WHP for milk and meat in Lao PDR. 10 | LENDZELE et al. 4 | D I S CU S S I O N Interestingly, the average lesion size in the control group increased as the trial progressed, suggesting that healing was compromised, This study reports the first field treatment trial using the novel topi- presumably from secondary bacterial infection. This may also explain cal anaesthetic wound formulation (Tri-Solfen®) as a therapy for the the observation that in the MO-treated cohort, the average lesion clinical management of FMD in Africa. It compared the clinical ef- size decreased until day 12, where it was recorded to increase from ficacy of this approach with the commonly used parenteral antibi- 0.17 cm to 0.75 cm at day 15, possibly from the onset of secondary otic treatment for FMD (Moore-Oxy®) and animals that remained infection presumably following depletion of residual oxytetracyline. untreated. Despite necessary limitations on numbers of farmers However, differences in stage of infection may also have influenced and cattle recruited for the trial, due to low availability of resources, the study here, particularly as the average lesion size in the control the results obtained provided a clear indication of the therapeutic cohort was 0cm at trial on day 0, with vesicles yet to rupture. advantage for TS for FMD, as recently described in Laos (Windsor The farmer observations of clinical impacts also provided inter- et al., 2020). Importantly, all participants considered it was very esting results which could be used as animal welfare indicators as successful trial, with high levels of

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ntly, all participants considered it was very esting results which could be used as animal welfare indicators as successful trial, with high levels of appreciation by participants for well as indicators of clinical response to the treatments. The mo- their involvement, the product examined (TS) and the clear clinical bility of all 12 cattle in the TS-treated cohort returned immediately, responses observed. suggesting this is a very useful therapy to enable cattle to walk and Lesion healing scores across the three groups revealed superior gain access to water and feed. In the MO-treated cohort, 10 of the results for the TS-treated cohort, with more rapid healing of cor- animals also immediately returned to walking. However, of the con- onary band lesions incurred from FMD. Although the MO-treated trol group, only five had immediate mobility and four animals did not cohort also achieved a reasonable score, the untreated control ani- return to walking even by day 15. There was one animal in the MO- mals had lesions persisting for in excess of 2 weeks, indicative of the treated cohort that did not return to mobility until day 12. In total, prolonged clinical course and debilitating impacts of FD on grazing 7/36 (19.4%) cattle had not returned to mobility by day 9, indicating animals. Although a subjective measurement, these scores provide that FMD is a severe disease as it renders animals immobile and that a useful indication of the healing rate of FMD lesions in field condi- although FMD is considered a low mortality disease, the animal wel- tions in Cameroon. fare impacts are clearly considerable. Similarly, appetite scores were highest in the TS-treated cohort, As oral vesicular lesions can have significant impacts on animal be- indicating that the treatment supported

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es were highest in the TS-treated cohort, As oral vesicular lesions can have significant impacts on animal be- indicating that the treatment supported the rapid return of appetite, haviours, recording the number of days the animals were anorexic was presumably reflecting the impact of oral anaesthesia enabling ani- considered important. In total, 11/12 cattle in the TS-treated cohort mals to eat shortly after treatment. After 3 days, it appeared that oral were reported to be eating the same day as treatment (day 0). This pain for the TS-treated cohort may have returned, although the re- compared to 9/12 cattle in the MO-treated cohort, with only 2/12 turn of appetite by day 6 suggests more rapid healing of oral lesions in the control cohort, with 9/12 of these untreated cattle remaining than untreated animals, as previously reported at 5 days (Windsor anorexic until day 15. Further, salivation is a common clinical sign with et al., 2020). Superior scores in this group were recorded at each cattle with vesicular disease, considered an overt indicator of oral data collection between day 6 and day 15, with the cohort receiving lesions and presumably oral pain. In all three cohorts, the majority treatment with MO also having reasonable appetite scores between of cattle had ceased salivating at day 0, with the TS-treated cohort days 6 and 15. The control cohort cattle had poor scores throughout, achieving 11/12, MO-treated cohort 10/12 and the controls 8/12. indicating that appetite remains poor for in excess of 2 weeks when Farmer reports of visible lameness of trial cattle indicated that FMD remains untreated. These findings suggest the possibility that 9/12 cattle in the TS-treated cohort ceased any lameness at day 0, secondary bacterial infections may have compromised

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gs suggest the possibility that 9/12 cattle in the TS-treated cohort ceased any lameness at day 0, secondary bacterial infections may have compromised the healing compared to 5/12 in the MO-treated cohort and 3/12 in the con- of oral lesions, with the antiseptic (cetrimide) and low pH properties trols. By day 6, no further cattle in the TS-treated cohort showed of TS, and the presence of circulating antibiotic of MO, ameliorating any lameness compared to 5/12 cattle in the MO-treated cohort still this. showing lameness by days 12 and 15, with lameness only ceasing Lesion size was measured (in cm) at six data collection intervals, by day 15 in 9/12 control cattle. For the question of how many days with the FMD-infected cattle lesion size decreasing rapidly in the prior to cattle returning to grazing, mixed results were recorded. In TS-treated cohort. By day 6, this cohort was performing well with the control cohort, 9/12 were reported grazing by day 0, with 8/12 average lesion size at 0.33 cm, despite commencing in the trial with in the TS-treated cohort returned to grazing by day 3 and all 12/12 the highest average lesion size. By day 9, the lesions in the TS-treated in the MO-treated cohort returning to grazing by day 6. TS has pre- cohort had almost entirely disappeared, remaining at 0 cm through viously been shown to be efficacious in controlling pain during the day 12 and 15. This is consistent with recent findings that healing of surgical treatment of hoof lesions in cattle (Stilwell et al., 2019). FMD lesions (Windsor et al., 2020) and induced husbandry wounds When asked for a broad overview of treatments applied to FMD, (Ferrer et al., 2020; Roberts & Windsor, 2019) is enhanced following six options were provided, including antibiotic formulations, tradi-

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lied to FMD, (Ferrer et al., 2020; Roberts & Windsor, 2019) is enhanced following six options were provided, including antibiotic formulations, tradi- topical treatment with TS, despite likely depletion of the actives in tional drugs, insecticides and petrol. Without speculating on the the formulation. This may be attributable to inclusion of a gel ma- therapeutic potential of each treatment, it does indicate that farm- trix providing prolonged protection for lesion and wound recovery. ers and para-veterinarians are inclined to treat affected animals with LENDZELE et al. | 11 something. The data on costs of treatment suggest there are only of presentation of animals for treatment, potentially assisting at- minimal differences between treatments and that cost should not tempts to improve disease reporting, surveillance and vaccination be an impediment to the changing of treatment choice. TS offers and biosecurity awareness through extension advice. It is con- a non-antimicrobial therapeutic option for treating clinical FMD, cluded that efforts to promote this new therapeutic approach to one that appears to have superior clinical efficacy to prolonged par- FMD management should be supported. enteral use of oxytetracylcine and potentially other antimicrobial therapies. As noted, use of TS potentially reduces the risk of AMR AC K N OW L E D G E M E N T S following FMD therapy (Windsor et al., 2020), yet as suggested in We thank the president of the cattle farmers and the sub-divisional these results, may also decrease putative secondary bacterial infec- delegates for livestock of Cameroon and particularly that of Tchabal tions occurring following the rupture of FMD vesicles. Of interest for support and help in informing farmers about this trial, and P.L.E.B was a recent

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ions occurring following the rupture of FMD vesicles. Of interest for support and help in informing farmers about this trial, and P.L.E.B was a recent observation that a lower rate of secondary infection for financial support. We also thank the ProgOncho field laboratory occurred following application of TS during surgical tail-docking for their cooperation and storage of samples, plus the driver and all of lambs, enabling the consideration of replacing routine antibiotic team members for their punctuality and time dedicated to this work. cover with a topical anaesthetic and antiseptic wound formulation The support of Medical/Animal Ethics Company for provision of the (Ferrer et al., 2020). product is gratefully acknowledged. Field studies involving animal treatments with assessments of clin- ical impacts are challenging and subject to potential bias, reflected in C O N FL I C T O F I N T E R E S T the limited published literature on FMD therapy. However, the results The authors report no conflicts of interest in this work. Studies obtained in this study demonstrate the superior clinical efficacy of a evaluating Tri-Solfen® and other therapies for aversive animal hus- single application to FMD lesions of the TS topical anaesthetic wound bandry interventions occurring prior to this study were funded by formulation. The participating farmers reported a 100% appreciation an Australian Research Council Linkage Grant from the Australian for the product in the treatment of FMD and expressed they were government with financial contributions from Animal Ethics Pty Ltd happy to have this product available for use in the region. These find- Australia and Bayer Animal Health Australia. However, this current ings were consistent with those from a recently reported clinical in-

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. These find- Australia and Bayer Animal Health Australia. However, this current ings were consistent with those from a recently reported clinical in- study did not receive funding from either of these companies, nor vestigation of TS therapy for FMD in Laos (Windsor et al., 2020) and did they have a role in study design, data collection and analysis, reports of the use of this product for FMD in other countries in Africa, decision to publish or preparation of the manuscript. including Niger, Nigeria and Kenya. It was recently suggested that TS may be viricidal against FMDVs if applied prior to or at the time of le- AU T H O R C O N T R I B U T I O N S sion rupture, potentially limiting virus transmission during FMD out- PW and SL designed the study, and PW, JY and PT provided analyti- breaks (Windsor et al., 2020). TS has a pH of 2.7–2.9 that is potentially cal and writing support. SL with support from JM, KB, KA and SD, sufficient to destroy FMDVs, plus a lidocaine concentration likely to performed the field trial activities. PT conducted the statistical anal- be directly viricidal against FMDVs (Haines et al., 1986). Further, TS yses and all authors contributed to the final draft of the manuscript. has recently been shown to reduce viral load in cutaneous lesions in sheep caused by the Orf virus (PW, unpublished observations). This E T H I C A L A P P R OVA L suggests that adoption of TS therapy for FMD may reduce disease The authors confirm that the ethical policies of the journal as transmission, the extent of animal suffering, plus rural household and noted on the authors guidelines page have been adhered to. In national socioeconomic losses in developing countries. addition to following current procedures on animal and human This study in Cameroon provides the

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ational socioeconomic losses in developing countries. addition to following current procedures on animal and human This study in Cameroon provides the quantitative assessment ethics processes in Cameroon, the authors communicated with confirming that TS is efficacious in hastening clinical recoveries, im- their Australian collaborators to ensure they complied with the mediately addressing pain and invoking more rapid healing of FMD National Health and Medical Research Council's (NHMRC) National lesions, as observed qualitatively in Laos (Windsor et al., 2020). Statement on Ethical Conduct in Human Research (2007) and the As clinical observations of improved animal welfare and enthu- Universities Australia Australian Code for the Responsible Conduct siastic feedback from farmers following TS therapy in Laos were of Research. This included ensuring that all participants provided confirmed in Cameroon, authorities proceeded with registration verbal informed consent for the collection of animal blood samples, of the product in Laos, with registration in Cameroon and several tissues, farmer interviews and participation in videos and images, other countries pending. These studies demonstrate that large ru- where written consent was unavailable due to farmer illiteracy. minants affected by FMD and treated with TS exhibit reduced pain, with reductions in both time to recovery and negative productiv- DATA AVA I L A B I L I T Y S TAT E M E N T ity impacts. TS therapy imposes no additional financial burden on The data that support the findings of this study are available from farmers and has the potential to replace antibiotics for treatment the corresponding author upon reasonable request. of a viral disease, reducing risks of AMR and residues in the food chain. As farmers in

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or treatment the corresponding author upon reasonable request. of a viral disease, reducing risks of AMR and residues in the food chain. As farmers in developing countries prioritize therapeutic in- ORCID terventions in FMD outbreaks, TS may also increase the likelihood Peter A. Windsor https://orcid.org/0000-0001-5629-3517 12 | LENDZELE et al. REFERENCES Hervé, M. (2020). RVAideMemoire: testing and plotting procedures for biostatistics. R package version 0.9-77. https://CRAN.R-proje​c t.org/ Agresti, A. (2002). Categorical data analysis (2nd edn.). John Wiley & Sons. packa​ge=RVAid​eMemoire AL-Lethie, A., AL-Lethie, S. F., El-Hawari, K., El-Khabaz, A. S., Elmeligy, Lendzele, S. S., Abdoulmoumini, M., Marvoungou, M. J., Ikoum, D., E., Khalphallah, A., Usama, T., & Mahmoud, U. T. (2018). Evaluation Mohammadou, B., Oumarou, L., Hiol, V. D., Rodrigue, M. N., Zinga- of clinical recovery and healing of oral lesions by 3 different thera- Koumba, C. R., Acapovi-Yao, G. L., Simon, D., & Garabed, R. (2019). peutic regimens in cattle with foot and mouth disease (FMD). Assiut Serological Epidemiology of Foot-and-mouth Disease among Veterinary Medicine Journal, 64(156), 89–95. Sedentary Mixed-species Herds in Adamawa Region, Cameroon. Bates, D., Maechler, M., Bolker, B., & Walker, S. (2015). Fitting linear Journal of Advances in Microbiology, 17(2), 1–14. mixed-effects models using lme4. Journal of Statistical Software, Lendzele, S. S., Marvoungou, M. J., & Rodrigue, M. N. (2019). Veterinary 67(1), 1–48. https://doi.org/10.18637​/jss.v067.i01 pharmaceuticals sold in cattle markets for the management of foot- Bertram, M. R., Delgado, A., Pauszek, S. J., Smoliga, G. R., Brito, B., and-mouth disease and flies in vina division (Adamawa-Cameroon). Stenfeldt, C., Hartwig,

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