Fasciola hepatica antioxidant and protease-inhibitor cocktail recombinant vaccines administered five times elicit potent and sustained immune responses in sheep but do not confer protection.

Our laboratory's vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite's ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60 F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power.

Herminthology: promoting gender equity in science and parasitology.

Gender inequity in Science, Technology, Engineering, and Medicine (STEM) fields, including parasitology, continues to limit the participation of women in scientific leadership and development. Here we highlight the aims and activities of Herminthology, an initiative promoting the work of women in parasitology, alongside the current status quo of men and women scientists in the discipline.

Two Distinct Superoxidase Dismutases (SOD) Secreted by the Helminth Parasite Fasciola hepatica Play Roles in Defence against Metabolic and Host Immune Cell-Derived Reactive Oxygen Species (ROS) during Growth and Development.

The antioxidant superoxide dismutase (SOD) catalyses the dismutation of superoxide, a dangerous oxygen free radical, into hydrogen peroxide and molecular oxygen. Superoxide generation during the oxidative burst of the innate immune system is considered a key component of the host defence against invading pathogens. We demonstrate the presence and differential expression of two SODs in Fasciola hepatica, a leaderless cytosolic (FhSOD1) and an extracellular (FhSOD3) form containing a secretory signal peptide, suggesting that the parasites exploit these enzymes in distinct ways to counteract reactive oxygen species (ROS) produced by cellular metabolism and immune defences. Both enzymes are highly expressed by the infective newly excysted juvenile (NEJ) stages and are found in abundance in their excretory-secretory products (ES), but only FhSOD1 is present in adult ES, suggesting that the antioxidants have different functions and pathways of secretion, and are under separate temporal expression control during the migration, growth, and development of the parasite. Functionally, the recombinant FhSOD1 and FhSOD3 exhibit similar activity against superoxide to their mammalian counterparts. Confocal immuno-localisation studies demonstrated the presence of FhSOD1 and FhSOD3 on the NEJ tegument and parenchyma, supporting our suggestion that these enzymes are secreted during host invasion to protect the parasites from the harmful oxidative bursts produced by the activated innate immune response. By producing superoxide enzymatically in vitro, we were able to demonstrate robust killing of F. hepatica NEJ within 24 h post-excystment, and that the lethal effect of ROS was nullified with the addition of SOD and catalase (the antioxidant enzyme responsible for the dismutation of hydrogen peroxide, a by-product of the SOD reaction). This study further elucidates the mechanism by which F. hepatica protects against ROS derived from cellular metabolism and how the parasite could mitigate damage caused by the host's immune response to benefit its survival.

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.

Low exposure of urban dogs in metropolitan Sydney, Australia to Toxocara canis demonstrated by ELISA using T. canis excretory-secretory (E/S) larval antigens.

Canine roundworm, Toxocara canis, is considered ubiquitous but patent infections are rare in adult owned urban dogs. Hepato-pulmonary migration of T. canis is common in young dogs, but in adult dogs, the migration of T. canis is arrested in tissues and larvae are inhibited. During this somatic migration, T. canis release excretory-secretory (E/S) larval antigens against which the host mounts an immune response. Detection of anti-T. canis E/S immunoglobulins is considered a proxy for the presence of arrested somatic T. canis larvae. By screening several cohorts of dogs in New South Wales (NSW), Australia, we determined the seroprevalence of anti-T. canis E/S in urban owned dogs visiting a veterinary teaching hospital in Sydney to be 3.8 % (n = 53), which was significantly lower (two-proportion z-test, P < 0.05) than the seroprevalence in pet dogs in regional western NSW (22.2 %, n = 63), and rehomed greyhounds (53.6 %, n = 28). Using a logistic regression model, the risk of testing positive in regional pet dogs (odds ratio [OR] = 37.0) and rehomed greyhounds (OR = 81.0) was significantly higher than in urban dogs (P < 0.05). Although routine deworming of dogs eliminates patent infection, our data show a low number of urban dogs with anti-T. canis E/S antibodies, which implies that the majority of these dogs were not exposed to T. canis previously, do not possess inhibited T. canis larvae, and in the case of intact females, will not transmit it to their puppies.

Pathogenicity and virulence of the liver flukes Fasciola hepatica and FasciolaGigantica that cause the zoonosis Fasciolosis.

Fasciolosis caused by the liver flukes Fasciola hepatica and Fasciola gigantica is one of the most important neglected parasitic diseases of humans and animals. The ability of the parasites to infect and multiply in their intermediate snail hosts, and their adaptation to a wide variety of mammalian definitive hosts contribute to their high transmissibility and distribution. Within the mammalian host, the trauma caused by the immature flukes burrowing through the liver parenchyma is associated with most of the pathogenesis. Similarly, the feeding activity and the physical presence of large flukes in the bile ducts can lead to anemia, inflammation, obstruction and cholangitis. The high frequency of non-synonymous polymorphisms found in Fasciola spp. genes allows for adaptation and invasion of a broad range of hosts. This is also facilitated by parasite's excretory-secretory (ES) molecules that mediate physiological changes that allows their establishment within the host. ES contains cathepsin peptidases that aid parasite invasion by degrading collagen and fibronectin. In the bile ducts, cathepsin-L is critical to hemoglobin digestion during feeding activities. Other molecules (peroxiredoxin, cathepsin-L and Kunitz-type inhibitor) stimulate a strong immune response polarized toward a Treg/Th2 phenotype that favors fluke's survival. Helminth defense molecule, fatty acid binding proteins, Fasciola-specific glycans and miRNAs modulate host pro-inflammatory responses, while antioxidant scavenger enzymes work in an orchestrated way to deter host oxidant-mediated damage. Combining these strategies Fasciola spp. survive for decades within their mammalian host, where they reproduce and spread to become one of the most widespread zoonotic worm parasites in the world.

Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia.

BACKGROUND: Canine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity-and thus disease-in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. METHODS: Blood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott's test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. RESULTS: A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9-15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen's kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26-13.51). All samples positive for the modified Knott's test were also positive for D. immitis DNA by PCR. CONCLUSIONS: This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.

Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1.

Trematode parasites of the genus Fasciola are the cause of liver fluke disease (fasciolosis) in humans and their livestock. Infection of the host involves invasion through the intestinal wall followed by migration in the liver that results in extensive damage, before the parasite settles as a mature egg-laying adult in the bile ducts. Genomic and transcriptomic studies revealed that increased metabolic stress during the rapid growth and development of F. hepatica is balanced with the up-regulation of the thiol-independent antioxidant system. In this cascade system thioredoxin/glutathione reductase (TGR) reduces thioredoxin (Trx), which then reduces and activates peroxiredoxin (Prx), whose major function is to protect cells against the damaging hydrogen peroxide free radicals. F. hepatica expresses a single TGR, three Trx and three Prx genes; however, the transcriptional expression of Trx1 and Prx1 far out-weighs (>50-fold) other members of their family, and both are major components of the parasite secretome. While Prx1 possesses a leader signal peptide that directs its secretion through the classical pathway and explains why this enzyme is found freely soluble in the secretome, Trx1 lacks a leader peptide and is secreted via an alternative pathway that packages the majority of this enzyme into extracellular vesicles (EVs). Here we propose that F. hepatica Prx1 and Trx1 do not function as part of the parasite's stress-inducible thiol-dependant cascade, but play autonomous roles in defence against the general anti-pathogen oxidative burst by innate immune cells, in the modulation of host immune responses and regulation of inflammation.

Fasciola Species Introgression: Just a Fluke or Something More?

The threats posed by a range of viral and bacterial zoonotic diseases inevitably receive renewed attention in the wake of global pandemic events due to their overt and devastating impacts on human health and the economy. Parasitic zoonoses, however, many of which affect millions of people each day, are frequently ignored. In the case of fasciolosis, caused by infection with Fasciola hepatica or Fasciola gigantica, this oversight has allowed for the expansion of areas of parasite sympatry and thus increased the incidence of hybridization and possible introgression betweenthe two species. Here we highlight how an increased demand for animal-derived protein, combined with a lack of appropriate tools for detection of these events, is changing the status quo of these zoonotic parasites.

What the fox? Cryptic Eucoleus [Capillaria] sp. in the respiratory tract of a cat from Australia.

Eucoleus aerophilus (syn. Capillaria aerophila) is a zoonotic trichuroid nematode parasite of dogs, cats and wild carnivores with a global distribution. The main reservoir species in Europe is the red fox, where it has been detected in up to 97% of animals surveyed. Despite the burgeoning feral cat and fox population in Australia, there is a paucity of information about the occurrence and molecular identity of E. aerophilus in these species. The occurrence of a gravid capillariid nematode in the bronchoalveolar lavage of a 12-week-old kitten from central New South Wales (NSW), with a history of lower respiratory signs that had been non-responsive to treatment with metronidazole or amoxicillin-clavulanic acid, prompted a detailed morphological and molecular investigation into the identity of the parasite including the examination of opportunistically-collected red fox tracheas from the region. A combination of PCR and next-generation sequencing yielded the first complete mitochondrial genome of E. aerophilus, collected from the red foxes in Australia, and revealed the presence of a cryptic Eucoleus [Capillaria] sp. in the kitten from central NSW. The protein-coding genes were 14-23% and 5-30% different (pairwise distance) at the nucleotide and amino acid sequences, respectively, which suggests the occurrence of a genetically distinct Eucoleus sp. lineage in Australia. The phylogenetic analysis using both Bayesian and the maximum likelihood methods demonstrated monophyly of the Trichuridae plus Capillariidae using amino acid sequences encoded by mitochondrial DNA. Analysis based on complete SSU rDNA sequences of Eucoleus [Capillaria] sp. and E. aerophilus placed them within Eucoleus spp. from the respiratory tract of their hosts. While Eucoleus spp. may not currently pose a significant threat to companion animals in Australia, their status as a recently emerged pathogen in Europe suggest that greater efforts should be made to understand the distribution and epidemiology of these parasites.

Using cerebrospinal fluid to confirm Angiostrongylus cantonensis as the cause of canine neuroangiostrongyliasis in Australia where A. cantonensis and Angiostrongylus mackerrasae co-exist.

Both Angiostrongylus cantonensis and Angiostrongylus mackerrasae have been identified along the east coast of Australia. A lack of A. mackerrasae genomic data until 2019, however, has precluded the unequivocal identification of the Angiostrongylus species responsible for neuroangiostrongyliasis in accidental hosts such as dog and man. The availability of a whole-genome data for A. mackerrasae, including mtDNA and ITS2 rDNA, enables discrimination of A. cantonensis from A. mackerrasae. The aim of this study was to develop diagnostic PCR assays to determine the species of Angiostrongylus based on the detection of Angiostrongylus DNA sequences in the cerebrospinal fluid (CSF) of canine patients with eosinophilic meningitis. An in silico workflow utilising available cytochrome c oxidase 1 (cox1) primers streamlined the laboratory work into empirical steps, allowing optimisation and selection of a PCR assay that met the required criteria for discrimination of A. cantonensis and A. mackerrasae DNA in low-template CSF samples. The adopted cox1 qPCR assay specifically amplified and enabled the differentiation of A. cantonensis from A. mackerrasae DNA and confirmed the presence of A. cantonensis DNA in 11/50 archived CSF samples. The DNA sequences demonstrated the presence of two distinct A. cantonensis cox1 haplotypes in dogs from eastern Australia. Species identification was further confirmed via the adoption of an ITS2 rDNA assay, providing confirmation of only A. cantonensis ITS2 rDNA in the CSF samples. To our knowledge, this is the first study to unequivocally demonstrate the antemortem presence of A. cantonensis DNA in CSF from clinically affected dogs. The study confirmed the long-held assumption that A. cantonensis is the causal agent of neuroangiostrongyliasis but refutes the dogma that there was a single introduction of A. cantonensis into Australia by the demonstration of two distinct A. cantonensis cox1 haplotypes.

Not gone but forgotten: Tritrichomonas foetus in extensively-managed bulls from Australia's Northern Territory.

Bovine trichomonosis, caused by infection with the protozoan parasite Tritrichomonas foetus, is globally recognised as a cause of reproductive failure in cattle. Maintained in clinically normal bulls, T. foetus infection results in infertility and abortion in infected cows. In Australia's Northern Territory (NT), logistical limitations associated with extensive livestock production inhibit wide-scale testing and diagnosis, allowing the parasite to persist undetected. In the present study, T. foetus was detected in 18/109 preputial cultures collected from bulls on a property in the NT with a history of low birth rates and reproductive failure using real-time PCR testing. Of the T. foetus-positive samples, 13/18 were genotyped using the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S rDNA unit. Selected samples were further characterised using the protein-coding genes of cysteine proteases (CP-1, 2, 4-9) and cytosolic malate dehydrogenase 1 (MDH-1) to determine if the isolates were 'bovine', 'feline' or 'Southern Africa' genotypes. All samples were 100% identical to the T. foetus 'bovine' genotype across all markers. This is the first reported case of trichomonosis in Australian cattle since 1988 and is a reminder that T. foetus should be considered whenever reproductive failure occurs in extensive cattle systems.

Which species is in the faeces at a time of global livestock movements: single nucleotide polymorphism genotyping assays for the differentiation of Fasciola spp.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a globally distributed zoonotic disease of livestock. While F. hepatica and F. gigantica have temperate and tropical distributions, respectively, parasite sympatry occurs in parts of Asia and Africa. A growing protein demand has the potential to facilitate the translocation of parasites from endemic to non-endemic areas, via associated international livestock movements. Such is the case in Southeast Asia, where livestock trade from F. hepatica-endemic countries into China and Vietnam may account for detection of F. hepatica hybrid/introgressed forms. Of particular importance is Lao People's Democratic Republic, which acts as a major livestock thoroughfare for the region. Our ability to understand the impacts of livestock-associated Fasciola spp. movements on local animal and human health is hindered by a lack of ante-mortem diagnostic tools allowing species differentiation. Molecular tools have been developed for Fasciola spp. differentiation, however those rely on access to pure DNA from adult specimens, limiting their application to post-mortem use. Our aim was to detect and differentiate F. hepatica from the endemic F. gigantica in local smallholder cattle in a region of Southeast Asia with frequent livestock trafficking. To do this we designed and validated ante-mortem molecular assays for Fasciola spp. differentiation targeting single-nucleotide polymorphisms (SNPs) within ITS1 and lsrRNA. We then deployed these SNP genotyping assays to diagnose Fasciola spp. infection in 153 local cattle from 27 villages in Northern Laos. We demonstrate the presence of F. hepatica DNA, confirmed by qualitative Sanger and quantitative Illumina amplicon sequencing of ITS1 and lsrRNA, and highlight the shortfalls of Sanger sequencing for Fasciola spp. identification due to the preferential amplification of F. gigantica nucleotides in mixed DNA samples. The outlined protocol enables rapid surveillance of faecal samples for the presence of Fasciola species eggs, their co-infection and/or infection with F. hepatica/F. gigantica hybrids.

Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle.

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance.

Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia.

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

Addressing the constraints of Tritrichomonas foetus sample collection in remote areas: lyophilized modified Diamond's media as a substitute for liquid medium.

Bovine trichomoniasis is a notifiable, reproductive disease of cattle caused by the parasite Tritrichomonas foetus. Culturing with modified Diamond's medium (MDM) is required to increase the low number of organisms received from a preputial sample, but is limited in application to remote areas as it requires continuous cold chain storage. This study utilized lyophilization to sustain the viability of MDM during transport in lieu of a continuous cold chain. All lyophilized MDM was able to sustain T. foetus after storage for 42 days at 24 °C, and the results demonstrated that lyophilized MDM was equally as viable as refrigerated liquid MDM. Storage of lyophilized MDM at room temperature for 1 and 7 days did not impact T. foetus yield, both with and without exposure to light. A limitation of the lyophilized MDM was demonstrated with a significant decrease in T. foetus yield when the media was stored at 37 and 58 °C. The lyophilization of MDM provides a robust method of transporting and storing medium prior to reconstitution and inoculation, for use in T. foetus diagnosis and surveillance in remote areas.

A quick and simple benchtop vortex egg-disruption approach for the molecular diagnosis of Fasciola hepatica from ruminant faecal samples.

Commonly employed diagnostic methods for Fasciola spp., such as a traditional sedimentation and faecal egg count, or a commercially available coprological ELISA, have limitations in their sensitivity or ability to differentiate species. A reliable DNA isolation method coupled with real-time PCR addresses these issues by providing highly sensitive and quantitative molecular diagnosis from faecal samples. The current study evaluated a standard benchtop vortex for F. hepatica egg disruption in sheep and cattle faecal samples and determined the minimum faecal egg load required for a positive result from un-concentrated (raw) faecal samples. The minimum faecal egg load for a positive real-time PCR result from 150 mg raw faecal sample was 10 and 20 eggs per gram for sheep and cattle, respectively. No significant difference (P = 0.4467) between disruptions on a benchtop vortex for 5 or 10 min was observed when compared to 40 s of disruption at 6.0 m/s in a benchtop homogeniser.

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.