Use of a local anaesthetic and antiseptic wound formulation for the treatment of lambs naturally infected with Orf virus.

Contagious ecthyma (CE) is a worldwide highly contagious zoonotic viral skin disease of sheep and goats. Treatment for Orf virus (ORFV) infection usually involves topical and oral antibiotics. An anaesthetic and antiseptic topical gel (Multisolfen® or Tri-Solfen®; MS®, Medical Ethics, Australia) has been documented as an efficacious therapy for lesions from mucosal and epithelial viral infections in ruminants. The present study tested a new treatment protocol of MS® for CE therapy on-farm in 150 lambs naturally infected with ORFV. Lambs were divided into three cohorts of 50 lambs each (C, D and E). Cohort C was treated with MS® 3 times with an interval of 3 days between treatments, cohort D was treated daily with hypochlorous acid, whilst cohort E served as untreated controls. The lambs were examined clinically every two days, weight measured weekly, with whole blood and sterile swabs from ORFV lesions collected for haematological analysis and specific ORFV PCR. Cohort C presented fewer lambs displaying ORFV-associated lesions than other cohorts at different times of the experiment. Further, lesions treated with MS® were milder compared with other cohorts. However, following cessation of therapy, most of the lambs again developed ORFV-associated lesions. No differences between cohorts were observed in weight, haematological and PCR results. These findings suggest that topical treatment with MS® is effective for CE in field conditions, especially in the first stages of the clinical course, although treatment with MS® may need to be extended a minimum of 4 weeks.

Understanding ATP Binding to DosS Catalytic Domain with a Short ATP-Lid.

DosS is a heme-containing histidine kinase that triggers dormancy transformation inMycobacterium tuberculosis. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases reveals a short ATP-lid. This feature has been thought to block binding of ATP to DosS's CA domain in the absence of interactions with DosS's dimerization and histidine phospho-transfer (DHp) domain. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS. We show that the closed-lid conformation observed in crystal structures of DosS CA is caused by the presence of Zn2+ in the ATP binding pocket that coordinates with Glu537 on the ATP-lid. Furthermore, circular dichroism studies and comparisons of DosS CA's crystal structure with its AlphaFold model and homologous DesK reveal that residues 503-507 that appear as a random coil in the Zn2+-coordinated crystal structure are in fact part of the N-box α helix needed for efficient ATP binding. Such random-coil transformation of an N-box α helix turn and the closed-lid conformation are both artifacts arising from large millimolar Zn2+ concentrations used in DosS CA crystallization buffers. In contrast, in the absence of Zn2+, the short ATP-lid of DosS CA has significant conformational flexibility and can effectively bind AMP-PNP (Kd = 53 ± 13 µM), a non-hydrolyzable ATP analog. Furthermore, the nucleotide affinity remains unchanged when CA is conjugated to the DHp domain (Kd = 51 ± 6 µM). In all, our findings reveal that the short ATP-lid of DosS CA does not hinder ATP binding and provide insights that extend to 2988 homologous bacterial proteins containing such ATP-lids.

Use of a Local Anaesthetic/Antiseptic Formulation for the Treatment of Lambs Experimentally Infected with Orf Virus.

Contagious ecthyma is a highly transmissible eruptive viral disease of the skin and mucosa of sheep and goats distributed worldwide. The treatment of orf lesions is usually based on the use of antiseptics and antibiotics for the management of presumptive secondary infections, increasing risks of antimicrobial resistance. The wound dressing formulation Tri-Solfen® (TS) containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation has been demonstrated to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease virus infection and reduced the orf viral load in lambs. In the present study, experimental infection with the orf virus was conducted in 50 newborn lambs and 25 animals were treated after the presence of the first lesions with TS and repeated three days later. Daily clinical examination, haematological, serological, biomolecular and post-mortem analyses were conducted during 34 days after treatment. Results indicated that treatment had no effect on weight gain and clinical progression of the lesions. It was determined that seroconversion after experimental infection occurs 34 days after infection and suggested that the deep basal epithelial location of the orf lesions may have prevented the therapy from having altered the clinical course.

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.

Molecular engineering of biocatalysts has revolutionized complex synthetic chemistry and sustainable catalysis. Here, we show that it is also possible to use engineered biocatalysts to reprogram signal transduction in human cells. More specifically, we manipulate cellular hypoxia (low O2) signaling by engineering the gas-delivery tunnel of prolyl hydroxylase 2 (PHD2), an iron-dependent enzymatic O2 sensor. Using computational modeling and rational protein design techniques, we resolve PHD2's gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. Systematic modification of these residues open the constriction topology of PHD2's gas tunnel with the most effectively designed mutant displaying 11-fold enhanced hydroxylation efficiency. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T cells reveal significant reduction in the levels of hypoxia inducible factor (HIF-1α) even under hypoxic conditions. Our studies reveal that activated PHD2 mutants can reprogram downstream HIF pathways in cells to simulate physiological O2-like conditions despite extreme hypoxia and underscores the potential of engineered biocatalysts in controlling cellular function.

Understanding ATP binding to DosS catalytic domain with a short ATP-lid.

DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS's CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP (Kd = 53 ± 13 µM). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.

Provision of High-Quality Molasses Blocks to Improve Productivity and Address Greenhouse Gas Emissions from Smallholder Cattle and Buffalo: Studies from Lao PDR.

Large ruminant production in developing countries is inefficient with low growth rates and likely high greenhouse gas emissions per unit of meat or milk produced. Trials conducted in Lao PDR from 2017 to 2020, studied ad libitum supplementation for 12 weeks with 20 kg high-quality molasses nutrient blocks (Four Seasons Pty Ltd., Brisbane, Australia), that were either non-medicated; fenbendazole-medicated (Panacur100®, Coopers Australia, 5 g/kg); triclabendazole-medicated (Fasinex®, Novartis Australia, 5 g/kg or 10 g/kg, respectively); or formulated with urea (8% or 10% urea, respectively). Average daily gains were determined for access to all molasses blocks and compared with access to control blocks, no supplementation, or previously determined free-grazing baseline average daily gains (55−84 g in cattle; 92−106 g in buffalo). Productivity was significantly improved following access to all molasses blocks. Average daily gains following access to 8% urea and control blocks were calculated for three age cohorts of cattle: young calves <8 m (238−298 g), growing cattle (143−214 g) and lactating cows (179−191 g). Modelling using IPCC Inventory software model V 2.69 of published data demonstrated a conservative net abatement of 350 kg CO2e was achievable over a 200-day feeding period. An additional trial of Emissions control blocks (n = 200) distributed to farmers (n = 60) and two educational institutions were conducted. Consumption rates (156 g/day) and farmer and institutional acceptance of these blocks were similar to our published findings with other molasses blocks, confirming all formulations of blocks improved animal productivity and body condition score, with healthier animals that were easier to manage. Modelling of changes in greenhouse gas emissions intensity identified an abatement of 470 kg CO2e per Emissions control blocks consumed, delivering a total project emissions abatement of 94 t CO2e. Provision of high-quality molasses blocks significantly improved smallholder large ruminant productivity and addition of greenhouse gas reducing agents is likely to achieve impressive abatement of greenhouse gas emissions due to improved efficiency of rumen fermentation and productivity.

Role of Topical Anaesthesia in Pain Management of Farm Animals, a Changing Paradigm.

Field evidence indicates that livestock producers are motivated by access to products that readily deliver pain management during husbandry interventions and, more recently, viral epidermal infectious diseases, including FMD. There has been impressive adoption in Australia of a farmer-applied spray-on topical anaesthetic wound formulation (TAF; Tri-Solfen®, Medical Ethics, Australia), initially for managing pain of the breech modification 'mulesing' procedure that reduces susceptibility of sheep to flystrike. Over 120 million lambs have now received pain relief and cattle producers have commenced using the TAF for a range of husbandry procedures. This product has demonstrated efficacy for surgical castration and tail docking of lambs, surgical castration and dehorning of calves, surgical castration of piglets, debridement of lesions of the hoof for lame cattle and, importantly, treatment of clinical FMD lesions, including decubitus ulcerations occurring from prolonged recumbency. Multimodal use of an NSAID for improved pain management is advocated, particularly meloxicam, available by prescription from veterinarians for injection and as an oral formulation (Ilium Buccalgesic®, Troy Laboratories, Australia), with current work assessing the potential for prolonged delivery in molasses blocks. Increased use of TAF with NSAIDs significantly reduces pain and suffering in livestock, with enhanced healing of FMD lesions, reduced viral loads from Orf infections in lambs and diminished necessity of 'antibiotic cover', assisting antimicrobial-resistance (AMR) stewardship.

Anaemia in Lambs Caused by Mycoplasma ovis: Global and Australian Perspectives.

Mycoplasma ovis (formerly Eperythrozoon ovis) is a haemotropic parasitic bacterium found within erythrocytes and distributed widely in global sheep and goat production regions. M. ovis is transmitted by biting flies and by contaminated instruments, causing morbidity and mortalities from anaemia, usually within 6 weeks following blood-exposure procedures, particularly vaccination, castration, ear tagging, mulesing, and occasionally crutching and shearing. Affected animals develop haemolytic anaemia and may have jaundice, causing abattoir condemnations. The typical history, clinical and pathological findings, display of M. ovis in blood smears and/or by PCR is diagnostic, although immune responses deplete M. ovis from smears; hence, in-contact healthy animals should be examined. Differential diagnoses include haemonchosis, fasciolosis, malnutrition (copper or vitamin B12 deficiency), and plant toxicities. M. ovis parasitaemia may persist, with recrudescence following stressful events, although most older sheep remain immune. Human infections have been reported. Inadequate socioeconomic data present difficulties in assessing the impact of M. ovis on production and as antimicrobial therapy is ineffective, its control requires management practices that minimize the impact of invasive procedures in periods when risks of M. ovis transmission are more likely. Although considered an emerging pathogen, recent improvements in welfare attitudes and husbandry practices on Australian sheep farms may potentially limit the transmission of M. ovis.

Alleles of unc-33/CRMP exhibit defects during Caenorhabditis elegans epidermal morphogenesis.

BACKGROUND: Microtubule-associated proteins regulate the dynamics, organization, and function of microtubules, impacting a number of vital cellular processes. CRMPs have been shown to control microtubule assembly and axon outgrowth during neuronal differentiation. While many microtubule-associated proteins have been linked to roles in cell division and neuronal development, it is still unclear the complement that control the formation of parallel microtubule arrays in epithelial cells. RESULTS: Here we show through time-lapse DIC microscopy that Caenorhabditis elegans embryos homozygous for the weak loss-of-function allele unc-33(e204) progress more slowly through epidermal morphogenesis, while animals homozygous for strong loss-of-function alleles exhibit more embryonic lethality. Identification of two novel missense mutations in unc-33(e572), Val476Gly, and Ser731Thr, lead to computational approaches to determine the potential effects of these changes on UNC-33/CRMP structure. Molecular dynamics simulations show that for Asp389Asn and Arg502His, two other known missense mutations, local changes in protein-protein hydrogen bonding affect the stability of the protein. However, the Val476Gly/Ser731Thr combination does not alter the structure or energetics of UNC-33 drastically when compared to the wild-type protein. CONCLUSIONS: These results support a novel role for UNC-33/CRMP in C. elegans epidermal development and shed light on how individual amino acid changes cause a loss-of-function in UNC-33.

Corrigendum: Progress With Livestock Welfare in Extensive Production Systems: Lessons From Australia.

[This corrects the article DOI: 10.3389/fvets.2021.674482.].

Can improving animal health and biosecurity knowledge of para-veterinarians in Cambodia assist in addressing challenges in smallholder livestock farming?

Para-veterinarians are crucial in providing front-line veterinary care and advice to smallholder farmers in countries with under-resourced national veterinary services. Many of these individuals undergo basic training with minimal monitoring and evaluation of performance and knowledge levels. To identify the knowledge, attitudes and practices of Cambodian para-veterinarians a cross-sectional survey (n = 80) of Village Animal Health Workers (VAHWs) was conducted during August 2018 in the provinces of Tbong Khmum and Takeo in southern Cambodia. Descriptive statistics indicated that VAHWs had a high level (mean score 86%) of animal health and biosecurity knowledge. As the Royal Government of Cambodia continues to move towards commercial agricultural value chains, VAHWs are crucial to ensuring the safety and sustainability of these systems. Binomial logistic regression modelling found the length of employment (p = .003), reporting of pig diseases (p = .007), recommending the quarantine of newly purchased animals (p = .008), treatment of cases of haemorrhagic septicaemia with antibiotics (p = .032) and washing footwear with water when entering or leaving a farm (p = .008) were all positively associated with a higher (≥92%) animal health and biosecurity knowledge score. These results demonstrate the correlation of positive biosecurity and disease reporting behaviours with high levels of knowledge, providing evidence that improved training of VAHWs can ensure they remain important in supporting the under-resourced national veterinary services. Regular monitoring and evaluation of training programmes for VAHWs will likely further strengthen the livestock sector in Cambodia.

Foot-and-mouth disease seroprevalence and reporting behaviours in nine northern provinces in Lao PDR: The current situation and challenges for control.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is one of the most important of global livestock diseases, impacting even-toed ungulates and distributed mostly in less developed countries that are home to 75% of the human population. A progressive control pathway for FMD (PCP-FMD) was developed to assist countries to better manage FMD risks and progress towards control and eradication. This requires evidence of current FMD seroprevalence to enable the informed risk assessment and the disease control planning required to progress along the initial stages of the PCP-FMD. Despite Laos being an active member of the South East Asia and China FMD campaign, these data have been challenging to obtain. To address this gap, a cross-sectional seroprevalence study in nine northern Lao provinces was conducted in early 2019. The study sampled 602 large ruminants and 19 goats from 30 villages. Overall, the large ruminant seroprevalence was 43.7% (95% CI 39.7-47.8), and 90% of sampled villages had at least one seropositive animal. Large ruminants suspected of having had clinical FMD in their lifetime were found to have an increased risk of being seropositive (odds ratio 1.96, 95% CI 1.06-3.65, p = .03). An examination of current reporting behaviours of farmers, village veterinary workers and district and provincial officials found that individuals were using their discretion as to whether disease events required reporting along the chain of command. This, plus the lack of a computerized reporting system, contributed to a loss of historical data, potentially compromising progression on the PCP-FMD. Laos requires ongoing support from donor agencies to improve the current animal disease surveillance system and implement effective FMD control strategies that can enable progress on the PCP-FMD.

Changes in farmer animal health and biosecurity knowledge, attitudes and practices: Insights from Cambodia and Laos.

Livestock agriculture in Cambodia and Laos is severely affected by endemic and exotic transboundary animal diseases, impacting household livelihoods and food and nutritional security. Collaborative animal health and biosecurity projects were conducted in each country between 2015 and 2019 aimed at increasing smallholder livestock production through mainly knowledge-based interventions in large ruminant disease prevention, nutrition, reproduction and marketing access. This study's objectives were to identify baseline animal health and biosecurity knowledge, attitudes and practices (KAP) of farmers, and temporal changes in key attitudes and practices associated with improved knowledge. Data for each country were analysed separately, and a formal between-country comparison was avoided due to major implementation and management differences. Binomial logistic regression without random terms compared the overall knowledge scores between 2015 and 2018 in each country, and binomial logistic regression with random terms was used to investigate possible associations between animal health and biosecurity knowledge scores and attitudes and practices. In Cambodia, the average animal health and biosecurity knowledge score increased from 4.8 (±2.2)/17 in 2015 to 13.0 (±2.8)/17 in 2018 (p < .0001). In Laos, the average scores improved from 7.2 (±2.5)/12 in 2015 to 7.9 (±2.9)/12 in 2018 (p = .0003). Clinical signs of foot-and-mouth disease were accurately reported by 100% of Cambodian and 88% of Lao project farmers, and overall, attitudes and practices improved in both countries over the study period. The belief that vaccination can improve livestock value was associated with increasing knowledge in both countries (Cambodia: OR 1.45, 95% CI 1.24-1.68, p < .0001; Laos: OR 1.43, 95% CI 1.16-1.75, p = .0006) as was the use of the services of village para-veterinarians if they had better training (Cambodia: OR 1.76, 95% CI 1.20-2.58, p = .004; Laos: OR 2.02, 95% CI 1.19-3.41, p = .009). However, the source of livestock information (District veterinary officials) was the only practice associated with knowledge in both Cambodia and Laos. The influence of resource scarcity on farmer decision making and knowledge measures was considered of particular relevance to the findings.

Knowledge, attitudes and practices of smallholder farmers on foot and mouth disease control in two Cambodian provinces.

Food-and-mouth disease (FMD) is endemic in Cambodia. The control programme for FMD has relied on vaccination, with poor vaccination uptake by smallholder farmers becoming an increasing concern. A study to improve the understanding of farmer knowledge, attitudes and practices of FMD control and vaccination was conducted in two Cambodian provinces (Kampong Cham and Pursat). The aim was to identify opportunities to improve the livestock disease control programmes provided by both the government and private sectors. The survey comprised 300 smallholder farmers using a one-on-one interview technique and was completed between January to February 2014. Results identified that over two-thirds of the respondent farmers had not vaccinated their cattle over 2 years (2011-2013). Of those who did, most cattle were vaccinated either once a year or once every 3 years. A booster had never been administered. It was concluded that the FMD vaccine had only been administered through an unreliable and limited government vaccination programme, and private FMD vaccination services were not accessed in the study areas. FMD outbreaks occurred every year during the study period, with a morbidity rate of over 30%. Isolation of first infected cattle from the household herd was not practiced, with treatment identified as the first preference intervention. Farmers often assisted other farmers to restrain and treat infected cattle both before (57%) and after (43%) their own cattle were infected. This indicated that most farmers did not practice basic biosecurity measures and chose to report FMD outbreaks to the village animal health workers (VAHW), friends, neighbours and relatives in preference to government officials. It was concluded that poor knowledge of disease transmission and biosecurity, with low FMD vaccination coverage and a focus on treatment, contribute to regular FMD outbreaks in these communities. Improvement of FMD control requires the cooperation of villagers, VAHWs and village leaders in disease reporting, with either improved funding of government vaccination services or establishing a private FMD vaccination service. Training programmes for farmers on disease transmission, and the importance of biosecurity and vaccination, including information on the cost-benefits of treatment versus full fee bi-annual FMD vaccination, are required.

Contributions of village animal health workers to foot-and-mouth disease control in Cambodia.

Local animal health services in rural communities in Cambodia are mainly provided by village animal health workers (VAHWs), although the participation and contribution of VAHWs to livestock disease prevention are uncertain. The participation of the VAHWs as identified by their 'dropout rate' was examined in a desktop review in December 2020 of the national data on VAHWs recorded between 2011 and 2020. The contribution and involvement of VAHWs in disease prevention programmes were examined in a survey conducted between February and March 2014, then analyzed in the context of other surveys of VAHW knowledge, attitudes and practices. The survey involved guided group discussion with VAHWs (n = 198) from the two Cambodian provinces of Kampong Cham and Pursat. This study identified that VAHWs generated less than 22% of their annual household incomes from animal health services. Less than one-third had vaccinated livestock against foot-and-mouth disease (FMD), with none having vaccinated cattle every 6 months during the study period, and nearly half of the VAHWs having never vaccinated their own cattle against FMD. As no privately provided FMD vaccination services occurred in these communities, with all vaccines delivered through the government-subsidized programme, the findings confirmed that VAHWs only vaccinated animals against FMD when vaccines were made available by the Government. The desktop review found that the number of VAHWs in 2020 declined by more than 24% since 2017, and the proportion of female VAHWs was consistently low, with a mean of 8.26 (±1.019). These findings confirm findings from previous studies that identified considerable weaknesses in the VAHW system in Cambodia, particularly in contributing to FMD control. Cambodian animal health authorities require more effective policies to strengthen the current VAHW system, improving their services delivery; their retention as 'active'; their development of more sustainable roles with lower 'dropout' rates and the prolonged gender inequity. With the limited availability of government-subsidized FMD vaccination currently, extension programmes that engage VAHWs and farmers in seeking privately funded and delivered FMD vaccination that incorporates appropriate multivalent FMD serotype vaccines of high quality, delivered in small dose vials from a robust cold chain, is suggested. This strategy would assist VAHWs to contribute to the provision of private livestock vaccination services that are likely essential for sustainable FMD prevention and control in Cambodia.

Are infectious reproductive pathogens of large ruminants a threat to improving food security? An investigation from Cambodia.

In Cambodia, information on common pathogens causing reproductive losses in cattle and buffalo are lacking, despite there being a need to address livestock health to enhance food security. We analysed stored buffalo (n = 29) and cattle (n = 471) serum samples collected in 2016 using commercially available enzyme-linked immunosorbent assay kits. Antibodies to Neospora caninum, bovine viral diarrhoea virus (BVDV), Leptospira interrogans serovar Hardjo and Brucella abortus were detected in buffalo samples at 79.3% (95% CI 64.6-94.0), 3.4% (95% CI 0-10.0), 0% and 0%, respectively, and in cattle at 4.2% (95% CI 2.4-6.0), 6.4% (95% CI 4.2-8.6), 8.1% (95% CI 5.6-10.6) and 0%, respectively. The high N. caninum seroprevalence in buffalo was associated with increasing age, with buffalo having a 13.1% chance of being seropositive at birth, increasing to 99.4% by age 7 (p = 0.045). This suggests a predominance of horizontal transmission, possibly from exposure to faeces from free-roaming village dogs. Cattle L. interrogans serovar Hardjo seroprevalence was highest in Tbong Khmum province (20.4%) compared to other provinces (p < 0.001), and may be compromising bovine fertility and creating a zoonotic risk for smallholders who may contract leptospirosis from farm work. These high infection rates prompt further research to determine: to what extent these pathogens are linked to the low reproductive efficiency observed in large ruminants in Cambodia, the risk factors to pathogen exposure and appropriate strategies to reduce these risks. Low BVDV and B. abortus exposure is an important observation. Increasing large ruminant livestock trade into the country will require improved biosecurity and disease surveillance to prevent their emergence. An enhanced understanding of the status of infectious reproductive livestock pathogens in Cambodia can assist development projects to make evidence-based strategies to enhance cattle and buffalo health and improve food security.

Progress With Livestock Welfare in Extensive Production Systems: Lessons From Australia.

The extensive livestock production industries are vital to the national economy of Australia. Continuing improvements to extensively-raised livestock welfare is desirable, necessary and in some situations mandatory, if the social license for animal sourced food and fiber production is to continue sustainably. However, meeting increasingly high welfare standards is challenging. The changing climate in this millennium, has seen the occurrence of two of the most severe drought periods on record in Australia, resulting in complex welfare issues arising from unforeseen disease, trade and environmental catastrophes. The onset of the first drought coincided with an uncontrolled epidemic of ovine paratuberculosis. It ended just prior to a temporary ban on live export of tropical cattle to Indonesia that induced a major market failure and led to severe morbidity and mortality on some beef properties. The second drought period progressed in severity and culminated in the most extreme bushfires recorded, causing unprecedented levels of mortality, morbidity and suffering in farmed animals and wildlife. Temperature extremes have also caused periodic heat-associated or cold-induced hyopthermia losses, requiring increased vigilance and careful management to reduce both temperature-induced stress during transport and the high ovine peri-parturient losses traditionally observed in extensive sheep farming. Several issues remain controversial, including surgical mulesing of wool sheep to manage flystrike, and the continuing live export trade of sheep and cattle. However, in reviewing the increasingly complex welfare challenges for the extensive livestock population industries that are export trade dependent and remain vulnerable to welfare activism, it appears progress has been made. These include development of prescribed livestock welfare Standards and Guidelines and the introduction of the Exporter Supply Chain Assurance System (ESCAS) to address export concerns. Further, the sheep mulesing crisis led to improved producer welfare attitudes and practices, including pain management during aversive husbandry procedures that is now occurring globally. Finally, innovations in animal welfare surveillance and assessment, are additional encouraging signs that suggest improving change management of extensive farm animal welfare is occurring that provides lessons well-beyond Australian shores.

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

INTRODUCTION: Orf is a highly contagious eruptive viral disease of the skin and mucosa of sheep and goats. Although vaccination with live or attenuated orf virus is the preferred option for disease control, the vaccine is unavailable in many countries. Treatment of orf lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was developed for pain relief in sheep undergoing surgical husbandry procedures in Australia. Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control. METHODS: One-month-old lambs (n=14), naturally infected with orf, were recruited from a farm during a natural outbreak of the disease. The animals were selected at the early stages of the infection and randomly divided into two cohorts: Group A (n=11) treated with the topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat embryonic fibroblasts (TIGEF). RESULTS: Although no significant differences were found in the clinical progression of the lesions and PCR quantification (p=0.722) between these small cohorts, there was 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.

Biflavonoid-Induced Disruption of Hydrogen Bonds Leads to Amyloid-ß Disaggregation.

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.