Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration.

African swine fever (ASF) is a transboundary viral disease which causes high mortality in pigs. In many low- and middle-income countries and in remote areas where diagnostic surveillance for ASF virus (ASFV) is undertaken, access to trained animal health technicians, sample collection, cold chain storage and transport of samples to suitably equipped laboratories can be limiting when traditional sampling and laboratory tests are used. Previously published studies have demonstrated that alternative sampling matrices such as swabs and filter papers can be tested using PCR without refrigeration for up to a week. This study used Genotube® swabs stored in temperate and tropical climates without refrigeration for four weeks after collection to demonstrate there was no change in test performance and results using loop-mediated isothermal amplification (LAMP) ASFV detection on a series of pig serum samples including serum spiked with a synthetic ASFV positive control, naturally acquired ASFV positive serum from Timor-Leste and negative ASFV serum samples. The use of Genotube® swabs for ASFV detection for surveillance purposes, coupled with testing platforms such as LAMP, can provide an alternative to traditional testing methodology where resources are limited and time from collection to testing of samples is prolonged.

Investigation of Weather Triggers Preceding Outbreaks of Acute Bovine Liver Disease in Australia.

Acute bovine liver disease (ABLD) is a hepatic disease affecting cattle sporadically in southern Australia, characterised histologically by striking periportal hepatocellular necrosis. The cause of ABLD is unknown; however, the seasonality and acute presentation of outbreaks suggest mycotoxin involvement. We described the geographical and seasonal occurrence of ABLD reports from 2010 to 2020 in Victoria, Australia, and explored potential weather triggers preceding 26 outbreaks occurring across 23 properties using a case-crossover design. Outbreaks occurred most frequently in autumn/early winter and in herds located along the southern coastal plain of Victoria, and occasionally within the low-lying regions of the Great Dividing Range. Lactating adult dairy cattle represented the most reported cases. We observed a significant association between an increase in average daily dewpoint in the 15 days preceding an ABLD outbreak, suggesting that dew formation may be a key determinant for this disease. Our findings support the etiology of a potent hepatotoxic agent that requires moisture for proliferation and/or toxin production.

Development of molecular detection methods of Bovicola ovis from sheep fleece.

The sheep body louse (Bovicola ovis) commonly referred to as sheep lice are small chewing ectoparasites of sheep. Infection results in significant economic costs to the Australian sheep industry due to reduced wool quality caused by chronic itching from sheep rubbing and biting fleece. Treatment relies on use of insecticides; however, resistance has developed against pyrethroid and other insect growth regulator lousicides. There is urgent need to develop cost-effective lice management to reduce the use of insecticides, with the application of insecticidal treatments only applied when an infestation is detected. However, the current detection method relies on fleece parting for detection of B. ovis which is highly dependent on the skill of the inspector, the number of sheep examined, and the prevalence and severity of the infestation. To improve B. ovis detection, a highly sensitive (5 × 10-8 ng/µL) and specific multiplex quantitative PCR which simultaneously detects sheep lice and sheep DNA was developed. In addition, a B. ovis loop-mediated isothermal amplification (LAMP) assay was developed for field use. The B. ovis LAMP (Bov-LAMP) assay was optimized to reliably detect B. ovis from wool samples down to 5 × 10-6 ng/µL, with time to positive (Tp) < 10 min. Both assays demonstrate high sensitivity and specificity, enabling rapid identification of B. ovis DNA from sheep fleece samples and have the capacity to be used for ongoing management and surveillance of B. ovis in Australian sheep flocks.

Use of Field Based Loop Mediated Isothermal Amplification (LAMP) Technology for a Prevalence Survey and Proof of Freedom Survey for African Swine Fever in Timor-Leste in 2019.

African Swine Fever (ASF) has been spreading in numerous southeast Asian countries since a major incursion in mainland China in 2018. Timor-Leste confirmed an outbreak of ASF in September 2019 which resulted in high mortalities in affected pigs. Pigs in Timor-Leste are the second most common type of livestock kept by villagers and represent a traditionally important source of income and prestige for householders. In order to understand the extent of ASF infected villages in Timor-Leste a prevalence survey was designed and conducted in November-December 2019. Timor-Leste has limited laboratory facilities and access to qPCR diagnostic tests. Therefore, a loop mediated isothermal amplification (LAMP) assay was used to detect ASF positive blood samples collected during the prevalence survey. The LAMP assay was proven to be a robust, highly specific and sensitive laboratory test for ASF suitable for use in the field and where there are limited laboratory facilities. The results of the prevalence survey allowed the extent of the ASF incursion to be delineated and the introduction of a disease response strategy to limit the spread of ASF and assist in the recovery of the pig population in Timor-Leste.

Analysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity.

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria. The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity. Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7-9 AM and 4-6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57). Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6-8.9 fold) and LFEC (5-16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control). The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %-90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes. In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

Fasciola hepatica Control Practices on a Sample of Dairy Farms in Victoria, Australia.

In Australia, little is known about the strategies used by farmers to control Fasciola hepatica (F. hepatica) infection in dairy cattle. Triclabendazole-resistant F. hepatica have recently been found on several dairy and beef properties in Australia. It is difficult to draw conclusions about how widespread resistance is in Australian dairy cattle because we have little information about flukicide usage, drug resistance testing, and alternative flukicide usage on-farm. The study objectives were to determine how dairy farmers are currently controlling F. hepatica and to identify knowledge gaps where F. hepatica control strategies need to be communicated to farmers to improve management. The survey was distributed online or by hard copy and 36 dairy farmers completed the survey. There were 34 questions including closed, open-ended, multicheck box, demographic, and text questions. Descriptive statistics were used to quantify each response. The survey results showed high use of clorsulon, limited rotation of flukicides, and limited use of diagnostic tests to inform treatment options and timing. There was poor adherence to best management practice in determining the dose of flukicides administered to cattle, with farmers often relying on estimating body weights or average body weights, suggesting that underdosing of animals is likely to be prevalent. Most respondents in this study did not isolate and quarantine treated and newly returned or purchased animals before joining them with the main herd. The research identified four knowledge gaps where communication needs to be enhanced to improve control of F. hepatica: diagnostic testing to inform flukicide use, rotation of flukicide actives, flukicide administration, and increased testing of replacement animals.

Clinical and pathologic features of acute bovine liver disease in Australia.

Acute bovine liver disease (ABLD) is a sporadic hepatic disease affecting cattle in southern Australia, characterized histologically by striking periportal hepatocellular necrosis. The cause of ABLD is unknown; however, the seasonality and acute presentation of outbreaks suggest mycotoxin involvement. We describe here the clinical and pathologic findings of ABLD in 45 naturally affected cattle from 13 outbreaks occurring from 2010 to 2019 in Victoria, Australia. Outbreaks occurred in herds located along the southern coastal plain of Victoria and were observed most frequently in lactating dairy cattle. Clinical signs commonly included a combination of mild photosensitization, progressive neurologic signs, and hypogalactia, which preceded death by ≤ 48 h. All affected animals had marked elevations in activities of glutamate dehydrogenase, aspartate aminotransferase, and gamma-glutamyl transferase. At autopsy, the most common lesions were serosal petechiae and/or gastrointestinal hemorrhage, and hepatomegaly with a pronounced hepatic reticular pattern. The principal histologic lesion was widespread-severe periportal hepatocellular coagulative necrosis and erythrocyte pooling-which often extended to massive necrosis. Lesions in other organs were uncommon. Our study of ABLD suggests involvement of a potent hepatotoxin that is either directly cytopathic or requires bioactivation by periportal-specific enzymes.

Towards understanding the liver fluke transmission dynamics on farms: Detection of liver fluke transmitting snail and liver fluke-specific environmental DNA in water samples from an irrigated dairy farm in Southeast Australia.

Livestock production around the world is impacted by liver fluke (Fasciola spp.) infection resulting in serious economic losses to the beef, dairy and sheep industries with significant losses of about $90 million per annum in Australia. Triclabendazole (TCBZ) is the most effective anthelmintic treatment available to control liver fluke infections; however, the widespread emergence of TCBZ resistance in livestock threatens liver fluke control. Alternative control measures to lower exposure of livestock to liver fluke infection would help to preserve the usefulness of current anthelmintic treatments. Environmental DNA (eDNA) based identification of liver fluke and the intermediate snail host in the water bodies is a robust method to assess the risk of liver fluke infection on farms. In this study, we used a multiplex quantitative PCR assay of water samples to detect and quantify eDNA of Fasciola hepatica (F. hepatica) and Austropeplea tomentosa (A. tomentosa), a crucial intermediate snail host for liver fluke transmission in South-east Australia. Water samples were collected from an irrigation channel for a period of 7 months in 2016 (February, March, May, September, October, November and December) at a dairy farm located at Maffra, Victoria, South-east Australia. Using an effective eDNA extraction method, the multiplex qPCR assay allows for the independent but simultaneous detection of eDNA released from liver fluke life stages and snails using specific primers and a probe targeting the ITS-2 region of the liver fluke and snail, respectively, with minimal inhibition from contaminants in field collected water samples. The sensitivity of this assay to detect eDNA of liver fluke and snails was observed to be 14 fg and 50 fg, respectively, in the presence of field collected water samples. Differential levels of liver fluke and snail specific eDNA in water were observed at the time points analysed in this study. The successful detection of eDNA specific to liver fluke and snails from the field collected water samples provides a precedent for the use of this method as a monitoring tool to determine the prevalence of liver fluke and liver fluke-transmitting snails in irrigation regions. Further, this method has the enormous potential to allow an assessment of the liver fluke transmission zones on farms and to inform the application of effective control strategies.

An idiopathic upper alimentary tract ulcerative syndrome in weaned dairy calves in Victoria, Australia.

An idiopathic clinical syndrome had been described in weaned dairy calves in the state of Victoria, Australia, where affected animals presented with diarrhoea, ill-thrift, enteritis and ulceration of the upper alimentary tract, with occasional oral/nasal ulcers. Between 7 November 2016 and 31 March 2019, 34 Victorian cattle herds were investigated, after each reported five or more weaned calves with diarrhoea and/or ill-thrift, or at least one calf with oral/nasal ulceration. Primary study objectives included the development of a detailed case definition for the clinical syndrome, termed upper alimentary tract ulcerative syndrome (UAUS) and the identification of potential causative virus(es) using metagenomics. A diagnosis of UAUS could not be made based solely on clinical signs and required histopathological assessment of post-mortem samples. Specifically, this included the identification of multifocal to coalescing areas of mucosal epithelial necrosis at all depths of the stratified squamous epithelium of the oesophagus, along with exclusion of bovine viral diarrhoea virus. Based on this case definition, twelve herds were diagnosed with clinical UAUS across the three dairying regions of Victoria, while thirteen were ruled UAUS-negative. The status of the nine remaining herds was unresolved due to a lack of required post-mortem samples. Metatranscriptomic analysis on oral swabs and oesopharyngeal samples from confirmed UAUS cases did not detect a virus common to the cross-sectional sample collection.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Plant and Fungal Hepatotoxicities of Cattle in Australia, with a Focus on Minimally Understood Toxins.

Plant- and fungus-derived hepatotoxins are a major cause of disease and production losses in ruminants in Australia and around the world. Many are well studied and described in the literature; however, this is not the case for a number of hepatotoxicities with economic and animal welfare impacts, such as acute bovine liver disease (ABLD), brassica-associated liver disease (BALD) and Trema tomentosa, Argentipallium blandowskianum and Lythrum hyssopifolia toxicity. Additionally, significant overlap in the clinical presentation and pathology of these conditions can present a diagnostic challenge for veterinarians. This review summarizes the current and most recently published knowledge of common plant- and fungus-associated hepatotoxins affecting cattle in Australia, with a focus on the mechanisms of toxicity and distinguishing diagnostic features. Consolidation of the current understanding of hepatotoxic mechanisms in cattle provides insight into the potential mechanisms of lesser-known toxins, including cellular and subcellular targets and potential metabolic pathways. In the absence of specific etiological investigations, the study of epidemiological, clinical and pathological features of hepatotoxicity provides valuable insights into potential toxic mechanisms and is integral for the successful diagnosis and management of these conditions.

The Consequences of Stigma for Knowledge Production: Sheep Producers' Attitudes to Footrot Diagnostics and Control in Australia.

In Australia, there is documented confusion from producers around the clinical disease of footrot, and anecdotally, knowledge of what tools are available for the diagnosis and management of footrot. When discussing footrot with producers, the authors noted a hesitation to discuss, with denial often expressed. The disease can be debilitating, both on the sheep's welfare and the producer's well-being, as it is a very difficult disease to manage and eradicate. Gaining an understanding of producer perceptions of the disease may help ensure any future actions for management and control are in-line with those identified by producers. A combination of a web-based, and manually distributed surveys of 45 sheep producers was conducted. This included closed- and open-ended questions, multi check box, and Likert scales. Responses were quantified by descriptive statistics and a thematic analysis conducted of short answers. The results of this survey indicate satisfaction with footrot diagnostics is low, while satisfaction with control methods is high. There was also a poor general understanding of footrot as a disease, and a general distrust in peers when it comes to correct management of footrot. This research addresses a gap in the literature about how sociological conditions affect diagnosis and control of footrot disease. It provides three main recommendations-simplifying the diagnostic message, encouraging a culture of trust among sheep producers and increasing governmental support-as a way to tackle this problem.

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).

Determination of the prevalence and intensity of Fasciola hepatica infection in dairy cattle from six irrigation regions of Victoria, South-eastern Australia, further identifying significant triclabendazole resistance on three properties.

Fasciola hepatica (liver fluke) is a widespread parasite infection of livestock in Victoria, South-eastern Australia, where high rainfall and a mild climate is suitable for the main intermediate host Austropeplea tomentosa. The aims of this study were to quantify the prevalence and intensity of F. hepatica in dairy cattle in the irrigated dairy regions of Victoria and determine if triclabendazole resistance was present in infected herds. Cattle in 83 herds from the following six irrigation regions were tested for F. hepatica: Macalister Irrigation District (MID), Upper Murray (UM), Murray Valley (MV), Central Goulburn (CG), Torrumbarry (TIA) and Loddon Valley (LV). Twenty cattle from each herd were tested using the F. hepatica faecal egg count (FEC) as well as the coproantigen ELISA (cELISA). The mean individual animal true prevalence of F. hepatica across all regions was 39 % (95 % credible interval [CrI] 27%-51%) by FEC and 39 % (95 % CrI 27%-50%) by cELISA with the highest true prevalence (75-80 %) found in the MID. Our results show that 46 % of the herds that took part in this study were likely to experience fluke-associated production losses, based on observations that herd productivity is impaired when the true within-herd prevalence is > 25 %. Using the FEC and cELISA reduction tests, triclabendazole resistance was assessed on 3 herds in total (2 from the 83 in the study; and 1 separate herd that did not take part in the prevalence study) and resistance was confirmed in all 3 herds. This study has confirmed that F. hepatica is endemic in several dairy regions in Victoria: triclabendazole resistance may be contributing to the high prevalence in some herds. From our analysis, we estimate that the state-wide economic loss associated with fasciolosis is in the order of AUD 129 million (range AUD 38-193 million) per year or about AUD 50,000 (range AUD 15,000-75,000) per herd per year.

Evaluation of loop-mediated isothermal amplification (LAMP) assay for detection of aprV2 positive Dichelobacter nodosus in-field by secondary users.

OBJECTIVE: Dichelobacter nodosus is the primary aetiological agent of footrot in sheep. Ovine footrot causes considerable economic losses and substantial animal welfare issues in the Australian sheep industry. Current methods for detecting D. nodosus are difficult, laborious and time-consuming. Recently, we developed a robust LAMP assay (VDN LAMP) that was able to identify aprV2 positive D. nodosus in-field. A major advantage of LAMP technology is the ability of the assay to be performed by non-specialists with minimal training. We aimed to assess the performance of the VDN LAMP in-field in comparison to a laboratory-based aprV2/aprB2 rtPCR when used by secondary users after training by the authors. RESULTS: Two animal health officers (termed secondary users) from Department of Primary Industries and Regions, South Australia (PIRSA) were trained in the use of VDN LAMP, before carrying out in-field testing on several locations in South Australia. The performance of VDN LAMP assay by secondary user 1 was shown to successfully detect 73.91% (n = 53) aprV2 positive samples, while secondary user 2 detected 37.93% (n = 30) aprV2 positive samples. Overall, the ability to identify virulent D. nodosus by VDN LAMP by secondary users was mixed for various reasons, however, this could be rectified by additional training and commercial production of the LAMP kits to increase stability. We envisaged in the future VDN LAMP will able to be used by non-specialists to aid control programs.

Optimization of a Loop Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of Dichelobacter nodosus With aprV2 (VDN LAMP) in Victorian Sheep Flocks.

Dichelobacter nodosus is the primary etiological agent of footrot in sheep and has a variety of virulence factors. Of these, AprV2, an extracellular protease, has been shown to be capable of causing severe or "virulent" disease symptoms under the right conditions. Due to this, a loop-mediated isothermal amplification (LAMP) assay for the detection of aprV2-positive D. nodosus (VDN LAMP) was developed and evaluated for field use. A sample of 19 sheep flocks (309 sheep) in Victoria, Australia, were tested to determine the optimum conditions for in-field VDN LAMP assay use and sampling, for detecting aprV2-positive D. nodosus infected sheep. VDN LAMP performance was compared to a validated rtPCR that detects aprV2 and the benign strain counterpart, aprB2, using biologically duplicate samples to determine sensitivity and specificity. Flocks were sampled either in winter-spring (moist) or early summer (dry) conditions and had a range of clinical expressions of the disease ovine footrot. Variables considered for optimizing field performance were: sample collection method, sample preparation, clinical expression of disease, and nature of the feet when sampled (moist vs. dry, clean vs. soiled). The test was found to perform best when sheep were sampled with moist, clean feet, using a dry swab with the sample prepared in alkaline polyethylene glycol, pH 13.0, as the collection buffer. A sensitivity of 89% and specificity of 97% was seen when used in-field under these conditions, when compared to aprV2 detection by rtPCR, with "very good" agreement to rtPCR results. This study shows the VDN LAMP test is easy to use in-field to identify the presence of aprV2-positive D. nodosus in sheep flocks.

Bone marrow fat analysis as a diagnostic tool to document ante-mortem starvation.

Veterinary diagnostic clinicians are increasingly presented with emaciated animals involved in suspected neglect cases. A rise in public awareness and media attention towards animal welfare, combined with changes in legislation and a demand for a higher standard of evidence be presented in animal neglect cases submitted for prosecutions, have created a need for an objective measurement of starvation, particularly given the lack of quantitative assessments at post-mortem examinations. Bone marrow fat (BMF) is the final fat reserve to be mobilised for energy by a calorie-deprived animal during a state of emaciation. Percentage of BMF has been used to study starvation in several species and may provide an objective measure of ante-mortem body condition. This paper reviews the literature on the use of BMF analysis as a post-mortem diagnostic test for ante-mortem starvation. Beginning with a general overview of starvation and usual methods of assessment to describe animals in poor condition, the analysis of BMF is then introduced. Various methods of BMF analysis are discussed, as well as factors that influence the amount of BMF. This review also discusses the limitations of BMF analysis and makes suggestions where future research should be primarily focused.

The development and deployment of a field-based loop mediated isothermal amplification assay for virulent Dichelobacter nodosus detection on Australian sheep.

Dichelobacter nododus is the causative agent of footrot, a major disease of sheep that creates welfare concerns and large economic loss. The virulence of D. nododus depends on the presence of extracellular proteases, AprV2 and AprB2, which differ by one amino acid. Strains possessing AprV2 can cause clinically virulent disease, while AprB2 may cause clinically benign disease. Current methods for detecting D. nodosus are difficult, laborious and time consuming. New techniques capable of rapidly detecting and typing D. nodosus are needed to aid control programs. Molecular methods, like real-time polymerase chain reaction (rtPCR) can detect aprV2 and aprB2, however, this assay is not field-deployable and cannot support local decision-making during an outbreak. Here we present a field-based molecular assay for detecting aprV2, using loop mediated isothermal amplification (LAMP). The aprV2 LAMP (VDN LAMP) assay was optimised to reliably detect aprV2 from laboratory purified genomic (gDNA) of virulent D. nodosus down to 5x10(-3) ng µL-1, with time to positive (Tp) ≤ 16 minutes, while aprB2 was unreliably detected at 5 ng µL-1 from 16-20 minutes. The use of field collected samples that were rtPCR positive for aprB2 resulted in no amplification, while aprV2 positive field samples by VDN LAMP assay are defined as having Tps' of < 20 minutes and melting temperature between 88.0-88.9°C. When compared to rtPCR, the VDN LAMP was shown to have a diagnostic specificity of 100% and sensitivity of 83.33%. As proof of concept, the VDN LAMP was taken on farm, with all processing occurring in-field. The on farm VDN LAMP successfully detected 91.67% aprV2 positive samples, no aprB2 positive samples (n = 9) or D. nodosus negative (n = 23) samples, with a kappa agreement of 'almost perfect' to rtPCR. This highlights the potential of the assay to inform local treatment decisions for management.

Assessment of a rtPCR for the detection of virulent and benign Dichelobacter nodosus, the causative agent of ovine footrot, in Australia.

BACKGROUND: Ovine footrot is a highly contagious bacterial disease of sheep, costing the Australian sheep industry millions of dollars annually. Dichelobacter nodosus, the causative agent of footrot, is a gram-negative anaerobe classed into virulent and benign strains as determined by thermostability of their respective protesases. Current methods for detection of D. nodosus are difficult and time-consuming, however new molecular techniques capable of rapidly detecting and typing D. nodosus have been reported. RESULTS: A competitive real-time PCR (rtPCR) method, based on the ability to detect a 2 nucleotide difference in the aprV2 (virulent) and aprB2 (benign) extracellular protease gene has been tested on Australian samples for determining detection rates, along with clinically relevant cut-off values and performance in comparison to the traditional culturing methods. The rtPCR assay was found to have a specificity of 98.3% for virulent and 98.7% for benign detection from samples collected. Sheep with clinical signs of footrot showed a detection rate for virulent strains of 81.1% and for benign strains of 18.9%. A cut-off value of a Ct of 35 was found to be the most appropriate for use in Victoria for detection of sheep carrying virulent D. nodosus. CONCLUSIONS: In summary, the rtPCR assay is significantly more capable of detecting D. nodosus than culturing, while there is no significant difference seen in virotyping between the two methods.

Development of a multiplex quantitative PCR assay for detection and quantification of DNA from Fasciola hepatica and the intermediate snail host, Austropeplea tomentosa, in water samples.

Liver fluke (Fasciola hepatica) infection is an increasing threat to livestock production resulting in serious economic losses to the beef, dairy and sheep industries in Australia and globally. Triclabendazole (TCBZ) is the main drug used to control liver fluke infections in Australia and the widespread emergence of TCBZ resistance in cattle and sheep threatens liver fluke control. Alternative control measures to lower exposure of livestock to fluke infection would be useful to help preserve the usefulness of current chemical flukicides. Environmental DNA (eDNA) sampling methodology and associated molecular techniques are suited to rapidly assess the presence of pathogens on farms. In the present study, we developed a water sampling method in combination with a multiplex quantitative PCR assay to detect and quantify DNA of F. hepatica and Austropeplea tomentosa (A. tomentosa), a crucial intermediate snail host for liver fluke transmission in South-east Australia. The multiplex qPCR assay allows for the independent detection of F. hepatica and A. tomentosa DNA using specific primers and a probe targeting the ITS-2 region of the liver fluke or snail. The method allows the highly specific and sensitive (minimal DNA detection levels to 14-50 fg) detection of F. hepatica or A. tomentosa. The method allows the detection of both liver fluke and snail eDNA in water samples. The effective quantification of liver fluke and snail eDNA in water samples using this assay could potentially allow researchers to both identify and monitor F. hepatica transmission zones on farming properties in South-east Australia which will better inform control strategies, with potential application of the assay worldwide.