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.

Livestock and livelihoods of smallholder cattle-owning households in Cambodia: the contribution of on-farm and off-farm activities to income and food security.

The majority of smallholder farming households in Cambodia are rurally based and rely on agriculture to support their livelihoods. However, in recent years, growth in the agriculture sector has stagnated with farmers facing several challenges including declining prices for traditional crops and irregular rainfall patterns. This has led to a need for farmers to diversify income sources with livestock promoted as a more viable livelihood activity, particularly the raising of cattle and poultry. However, uncertain profitability of livestock activities is a common perception by smallholders, especially where animals have not been traditionally viewed as a primary income source. To address this, information is required which compares the income and expenses associated with livestock raising to other on-farm activities and off-farm sources. This study reports on a survey of livelihood survey of 17 male and 21 female representatives of 20 households owning cattle in Cambodia, comparing the associated income and expenses of the various livelihood activities in the 12-month period from January to December 2016. Combined total household income from both on-farm and off-farm sources ranged from USD875 to 17730 with an average of USD6779, representing 51% and 49% from on-farm and off-farm activities, respectively. Cattle raising was the most common source of on-farm income (85%), contributing USD1064 and representing 22% of total household income. General household expenses, such as food and transportation (including expenses associated with off-farm employment), represented the majority of total household expenses (79%). Gross profit calculations indicated higher values for pig raising (USD1841.79), cattle (USD950.80) and non-rice crops (USD884) whilst the highest gross margin value was recorded for cattle (89.33%) followed by vegetables (85.27%) and non-rice crops (83.08%). Whilst wet season and dry season rice returned a negative gross profit value of USD197.27 and USD90.60 on average per household, respectively, both were ranked as most important for household consumption, followed by poultry, providing the main source of energy (rice) and protein (chicken meat) and sustaining household food requirements. The study concludes that although smallholder cattle-owning households in Cambodia undertake a diverse range of on-farm activities, as cattle raising provides a superior income source due to higher returns and lower variable costs, it could be promoted as a preferred livelihood activity by agencies conducting rural extension activities. Although consideration of available labour and monetary funds to invest in cattle raising is required, it was observed that income from off-farm sources may prove beneficial in providing the additional monetary funds to support cattle-raising activities and assist in providing generally poor smallholder households with enhanced economic resilience.

Survey of smallholder beef cattle production systems in different agro-ecological zones of Cambodia.

A survey was conducted to better understand the contribution of farm productivity to rural household income and identify differences in production systems, feeding practices and development constraints to smallholder beef cattle producers in the four agro-ecological zones (AEZs) of Cambodia. A semi-structured questionnaire was used to interview 360 households in the four AEZs: I, the Great Lake Floodplain; II, the Mekong Floodplain; III, the Coastal and IV, the Plateau/Mountainous. In addition, samples of common nutritional resources used for cattle feed were collected for nutrient composition analysis, plus cattle were scored for body condition. Rice farming and cattle production were the most common sources of income in all AEZs. The average cattle herd size was 3.7 (SD = 2.4), but the majority of households raised 1-3 animals. The most common cattle management system was grazing with supplementation, mainly with rice straw and 'cut-and-carry' natural grasses fed during the wet season in all AEZs. The body condition score of all cattle types was 3.2 (SD = 0.8), except for cows in lactation that were 1.8. Major constraints to cattle production in AEZs I, II and III were lack of quality feed resources, capital for cattle production and concerns on breed quality, whereas in AEZ IV, diseases were identified as the main constraint. This survey confirms the importance of cattle to smallholders in the four AEZs. Interventions including farmer education to improve husbandry skills, increase the utilisation of forages and crop residues and address disease issues are necessary to enhance cattle production and rural livelihoods in Cambodia.

Improving smallholder food security through investigations of carcass composition and beef marketing of buffalo and cattle in northern Lao PDR.

This study determined the carcass composition of Lao indigenous buffalo (Bubalus bubalis) and cattle (Bos indicus), then examined trends in bovine meat marketing following review of records of beef production and prices in the two major cities of Luang Prabang (LPB) and Xieng Khoung (XK) provinces in northern Laos. Samples from 41 buffalo and 81 cattle (n = 122) were collected from animals slaughtered in May-June 2014, with live weights, carcass weights and other carcass-related variables collected. The animals were classified into four age cohort groups (<2, 2-<4, 4-6 and >6 years) with quantitative and dichotomous qualitative traits determined. There were significant differences in buffalo and cattle predicted mean carcass weights between age classification categories (p = 0.003 and 0.001) but not in dressing percentages (p = 0.1 and 0.1). The carcass weight of buffalo was 104 (±23.1)-176 (±12.0) kg compared to 65 (±8.7)-84 (±6.5) kg of cattle, with dressing percentages of 37-40 and 39-42 %, respectively. Despite an average bovine meat price increase of 42-48 % between 2011 and 2013, there was a reduction in the numbers of large ruminants slaughtered in the surveyed cities of LPB (11 %) and XK (7 %), with bovine meat availability per person of 5.2-6.6 kg (LPB) and 3.0-3.8 kg (XK). Improving the sustainability of the bovine meat supply in Laos requires a systems approach involving improvements to animal health and production, livestock marketing, plus the critical development of improved slaughterhouse facilities enabling a meat-processing sector to emerge. This development pathway is of particular importance for building the capacity of Laos to reduce food insecurity and alleviate the poverty of its largely rural smallholder community.

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.

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.

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

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

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.

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.

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.

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

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

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.

Impact of a Topical Anaesthesia Wound Management Formulation on Pain, Inflammation and Reduction of Secondary Infections after Tail Docking in Lambs.

We examined several procedures for surgical tail docking; with and without general anaesthesia (GA), including the use of a topical wound gel formulation to provide pain relief (PR) and improve healing after surgery, containing local anaesthetics lignocaine and bupivacaine, with cetrimide and adrenalin. Forty-four lambs were recruited into four equal cohorts: Groups A and C, the tail was excised with a scalpel without anaesthesia or stitches; Groups B and D, the tail was surgically excised and stitched under GA; Groups C and D wounds were immediately sprayed with PR. Behavioural observations identified that Groups A and C displayed significantly less pain-related behaviours than Groups B and D shortly after the procedure, especially if treated with PR. Similarly, the mean of days when animals showed no signs of wound infection was longer in the groups not undergoing stitching. Finally, treatment with PR appeared to reduce the cortisol response and avoided the elevation of serum amyloid A in lambs where the tail was excised without general anaesthesia. In conclusion, surgical tail-docking without GA but where wounds are immediately sprayed with PR, appears as an affordable and more welfare-appropriate method for conducting tail docking in lambs.

Effect of Lignocaine and a Topical Vapocoolant Spray on Pain Response during Surgical Castration of Beef Calves.

This study assessed the efficacy of pre-operative injected lignocaine and peri-operative topical vapocoolant spray, administered as most practical for incorporation into routine calf castration procedures. Calves were randomly allocated to: (1) sham castration (SHAM); (2) surgical castration (CAST); (3) surgical castration with lignocaine (LIG); and (4) surgical castration with vapocoolant spray (VAPO). Calf behavioural responses were scored at different stages of the sham castration or castration procedure. Maximum ocular temperatures were measured at three time-points relative to restraint and treatment. There were significant effects of treatment (p < 0.001) and stage of procedure (p < 0.001) on calf behavioural response. SHAM calves were more likely to display less severe responses compared to all other calves and LIG calves were more likely to display less severe responses compared to VAPO calves. Calves were more likely to display more severe responses to extrusion of the first spermatic cord compared to all other stages of castration, and to extrusion of the second spermatic cord compared to severing of the second spermatic cord. There was a significant effect of time (p < 0.001) on ocular temperature, with ocular temperature being greater following sham castration or castration. In this study, there was no evidence of pain reduction during castration of calves by either lignocaine or vapocoolant spray.

Evaluating treatments with topical anaesthetic and buccal meloxicam for pain and inflammation caused by amputation dehorning of calves.

To assess the effects of a topical anaesthetic (TA) and buccal meloxicam (BM) on behaviour, maximum wound temperature and wound morphology following amputation dehorning of beef calves, 50 unweaned Hereford calves were randomly allocated to: (1) sham dehorning / control (CON, n = 14); (2) amputation dehorning (D, n = 12); (3) amputation dehorning with pre-operative buccal meloxicam (DBM, n = 12); and (4) amputation dehorning with post-operative topical anaesthetic (DTA, n = 12). Videos of the calves were captured for 3 h following treatment. Each calf was later observed for 5 min every hour and the frequency and duration of specific behaviours displayed during these focal periods was recorded. Infrared and digital photographs of dehorning wounds were collected from all dehorned calves on days 1, 3 and 7 following treatment. Infrared photographs were used to identify the maximum temperature within the wound area. Digital photographs were used to score wounds based on visual signs of inflammation and healing, using a numerical rating scale of 1 to 3, with morphological aspects of inflammation increasing and morphological aspects of healing decreasing with progressive scores. CON calves displayed fewer head shakes than all dehorned calves at 2 and 3 h following treatment (P = 0.025). CON and DTA calves displayed less head turns than DBM calves at 2 h following treatment (P = 0.036). CON calves displayed fewer combined point behaviours than all dehorned calves at 2 h following treatment (P = 0.037). All dehorning wounds had a greater maximum temperature on days 3 and 7 compared to day 1 (P = 0.003). All wound morphology scores decreased from day 1 to day 3 and wound morphology scores of DBM and DTA calves increased from day 3 to day 7 (P = 0.03). Although flystrike may have confounded these observations, no clear effects of TA or BM on behaviour, maximum wound temperature or wound morphology following dehorning of calves were observed. Further research is required to evaluate the analgesic efficacy of these products for amputation dehorning of calves.

Comparison of early detection of Fasciola hepatica in experimentally infected Merino sheep by real-time PCR, coproantigen ELISA and sedimentation.

Fasciolosis due to infection with Fasciola hepatica, Fasciola gigantica or their hybrids is a significant global cause of livestock production loss. Infection is commonly diagnosed by a labour-intensive sedimentation and faecal egg count (FEC), which has limited throughput and is only applicable after completion of the 8-12 week pre-patent period (PPP). A commercially-available ELISA for the detection of coprological antigen (coproELISA) enables detection prior to the completion of the PPP and is suitable for diagnosis of larger sample sizes, although the sensitivity reported under experimental infection settings can be difficult to replicate in the field, particularly in cattle. A recently-published real-time PCR workflow for the sensitive detection of Fasciola spp. DNA in faecal samples provides increased sample throughput, although the point at which this technique is first able to diagnose infection remains unknown. Other tools for the molecular diagnosis of fasciolosis, such as conventional PCR and loop-mediated isothermal amplification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal samples were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. Samples were first considered positive at 6-8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of samples were positive by all three methods. To evaluate the capacity of the real-time PCR approach to detect infection prior to completion of the PPP, two methods of sample preparation were compared at 2 WPI: (i) 150 mg raw faecal samples and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of sample preparation yielded positive results at 2 WPI suggesting that DNA amplification by real-time PCR is associated with faecal egg load.

Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.

BACKGROUND: Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples. METHODOLOGY/PRINCIPAL FINDINGS: A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow. CONCLUSIONS/SIGNIFICANCE: Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.