Anoplocephalid tapeworms in mountain gorillas (Gorilla beringei beringei) inhabiting the Volcanoes National Park, Rwanda.

Cestodes of the family Anoplocephalidae parasitize a wide range of usually herbivorous hosts including e.g. rodents, ungulates, primates, elephants and hyraxes. While in some hosts, the epidemiology of the infection is well studied, information is lacking in others. In this study of mountain gorillas in the Virunga Massif, an extensive sample set comprising adult cestodes collected via necropsies, proglottids shed in feces, and finally, fecal samples from both night nests and identified individuals were analysed. Anoplocephala gorillae was the dominant cestode species detected in night nest samples and individually known gorillas, of which only 1 individual hosted a Bertiella sp. It was shown that the 2 species can be distinguished through microscopy based on egg morphology and polymerase chain reaction (PCR) assays for diagnostics of both species were provided. Sequences of mitochondrial (cox 1) and nuclear (ITS1, 18S rDNA, 28S rDNA) markers were used to evaluate the phylogenetic position of the 2 cestodes detected in mountain gorillas. Both types of fecal samples, from night nests and from identified individuals, provided comparable information about the prevalence of anoplocephalid cestodes, although the analysis of samples collected from identified gorilla individuals showed significant intra-individual fluctuation of A. gorillae egg shedding within a short period. Therefore, multiple samples should be examined to obtain reliable data for wildlife health management programmes, especially when application of anthelmintic treatment is considered. However, while A. gorillae is apparently a common symbiont of mountain gorillas, it does not seem to impair the health of its host.

Whole genome amplification and sequencing of individual Dirofilaria immitis microfilariae.

Dirofilaria immitis is a filarial parasitic nematode of veterinary significance. With the emergence of drug-resistant isolates in the USA, it is imperative to determine the likelihood of resistance occurring in other regions of the world. One approach is to conduct population genetic studies across an extensive geographical range, and to sequence the genomes of individual worms to understand genome-wide genetic variation associated with resistance. The immature life stages of D. immitis found in the host blood are more accessible and less invasive to sample compared to extracting adult stages from the host heart. To assess the use of immature life stages for population genetic analyses, we have performed whole genome amplification and whole-genome sequencing on nine (n = 9) individual D. immitis microfilaria samples isolated from dog blood. On average, less than 1% of mapped reads aligned to each D. immitis genome (nuclear, mitochondrial, and Wolbachia endosymbiont). For the dog genome, an average of over 99% of mapped reads aligned to the nuclear genome and less than 1% aligned to the mitochondrial genome. The average coverage for all D. immitis genomes and the dog nuclear genome was less than 1, while the dog mitochondrial genome had an average coverage of 2.87. The overwhelming proportion of sequencing reads mapping to the dog host genome can be attributed to residual dog blood cells in the microfilariae samples. These results demonstrate the challenges of conducting genome-wide studies on individual immature parasite life stages, particularly in the presence of extraneous host DNA.

Detection of tick-borne bacterial DNA (Rickettsia sp.) in reptile ticks Amblyomma moreliae from New South Wales, Australia.

Ticks are major arthropod vectors of disease, transmitting tick-borne pathogens during blood meal episodes. Rickettsia spp. and Borrelia spp. are two tick-borne pathogens of zoonotic concern previously identified in DNA isolates from the tick genera Amblyomma and Bothriocroton associated with reptilian hosts in Australia. Some reports suggest that these reptile ticks bite and attach to humans via accidental parasitism and transmit disease, with the tick Bothriocroton hydrosauri known to transmit Rickettsia honei or Flinders Island Spotted Fever Rickettsia to humans. This descriptive study aims to identify the ticks collected from wild reptiles submitted to veterinary clinics and captured by snake rescuers from New South Wales (NSW), Australia, and detect the presence of tick-borne bacterial DNA using quantitative polymerase chain reaction (qPCR) to detect Rickettsia spp. and Bartonella spp. and conventional nested-PCR to detect Borrelia spp. Morphological identification revealed ticks removed from one eastern blue-tongued lizard (Tiliqua scincoides scincoides) from North-Eastern NSW (Lismore), one eastern blue-tongued lizard from the Greater Sydney area (Canley Heights), one diamond python (Morelia spilota spilota) from the Greater Sydney area (Woronora Heights) and one red-bellied black snake (Pseudechis porphyriacus) from the Greater Sydney Area (Cronulla) in New South Wales were Amblyomma moreliae. No ticks were positive for Bartonella spp. and Borrelia spp. DNA using real-time PCR targeting ssrA gene and nested PCR targeting Borrelia-specific 16S rRNA gene, respectively. Real-time PCR targeting gltA, ompA, ompB and 17kDa gene of Rickettsia spp. revealed 14 out of 16 ticks were positive. The undescribed Rickettsia sp. DNA was identical to that previously recovered from reptile ticks in Australia and closely related to Rickettsia tamurae and Rickettsia monacensis, both of which are aetiologic pathogens of the Spotted Fever Group Rickettsiosis (SFGR). These results accentuate the ongoing need for increased study efforts to understand zoonotic potential of bacteria from reptile ticks and the tick-reptile-human relationship.

Human Neural Larva Migrans Caused by Ophidascaris robertsi Ascarid.

We describe a case in Australia of human neural larva migrans caused by the ascarid Ophidascaris robertsi, for which Australian carpet pythons are definitive hosts. We made the diagnosis after a live nematode was removed from the brain of a 64-year-old woman who was immunosuppressed for a hypereosinophilic syndrome diagnosed 12 months earlier.

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures.

Gastrointestinal nematodes threaten the productivity of grazing livestock and anthelmintic resistance has emerged globally. It is broadly understood that wild ruminants living in sympatry with livestock act as a positive source of refugia for anthelmintic-susceptible nematodes. However, they might also act as reservoirs of anthelmintic-resistant nematodes, contributing to the spread of anthelmintic resistance at a regional scale. Here, we sampled managed sheep and cattle together with feral goats within the same property in New South Wales, Australia. Internal transcribed spacer 2 (ITS-2) nemabiome metabarcoding identified 12 gastrointestinal nematodes (Cooperia oncophora, Cooperia punctata, Haemonchus contortus, Haemonchus placei, Nematodirus spathiger, Ostertagia ostertagi, Teladorsagia circumcincta, Oesophagostomum radiatum, Oesophagostomum venulosum, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus rugatus). Isotype-1 ß-tubulin metabarcoding targeting benzimidazole resistance polymorphisms identified 6 of these nematode species (C. oncophora, C. punctata, H. contortus, H. placei, O. ostertagi and T. circumcincta), with the remaining 3 genera unable to be identified to the species level (Nematodirus, Oesophagostomum, Trichostrongylus). Both ITS-2 and ß-tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta, known from domestic goats in France. Of the gastrointestinal nematodes detected via ß-tubulin metabarcoding, H. contortus, T. circumcincta, Nematodirus and Trichostrongylus exhibited the presence of at least one resistance genotype. We found that generalist gastrointestinal nematodes in untreated feral goats had a similarly high frequency of the benzimidazole-resistant F200Y polymorphism as those nematodes in sheep and cattle. This suggests cross-transmission and maintenance of the resistant genotype within the wild ruminant population, affirming that wild ruminants should be considered potential reservoirs of anthelmintic resistance.

Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum.

Cyst-forming coccidia, Toxoplasma gondii and Neospora caninum, are recognised as important causes of animal disease. Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect T. gondii and N. caninum while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. T. gondii and N. caninum. The T. gondii assay is based on a 529-bp repetitive (REP) element and the N. caninum assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either T. gondii or N. caninum. The T. gondii and N. caninum multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.

Sarcocystis sp. infection (Apicomplexa: Sarcocystidae) in invasive California kingsnake Lampropeltis californiae (Serpentes: Colubridae) in Gran Canaria.

Invasive species pose a threat not only to biodiversity because they displace or compete with native fauna, but also because of the pathogens they can host. The Canary Islands are an Atlantic biodiversity hotspot threatened by increasing numbers of invasive species, including the California kingsnake Lampropeltis californiae, which was recently introduced to Gran Canaria. Seventy-seven snakes were examined for gastrointestinal parasites in 2019­2020. Sporocysts of Sarcocystis sp. were detected in 10 of them; detection of gamogonia stages in histological sections of 3 snakes confirmed the snake as a definitive host. Partial ssrDNA was amplified using SarcoFext/SarcoRext primers; an additional sequence of Sarcocystis was obtained from the tail muscle of the endemic Gran Canaria giant lizard Gallotia stehlini for a comparison. Identical ssrDNA sequences of unknown Sarcocystis sp. were obtained from 5 different snakes. Phylogenetic analysis showed that Sarcocystis sp. isolated from invasive California kingsnakes is unrelated to Sarcocystis provisionally considered S. stehlini from the endemic lizard. The dixenous coccidia are rarely reported to invade new predator­prey systems. However, the present data suggest that previously unknown Sarcocystis sp. is circulating among invasive snakes and as yet unknown vertebrate intermediate hosts, with undetermined consequences for the Gran Canaria ecosystem.

Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.

BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Magnetic resonance imaging in dogs with neuroangiostrongyliasis (rat lungworm disease).

The magnetic resonance imaging (MRI) appearance of the brain and spinal cord in humans with neuroangiostrongyliasis (NA) due to Angiostrongylus cantonensis infection has been well reported. Equivalent studies in animals are lacking. This case series describes clinical and MRI findings in 11 dogs with presumptively or definitively diagnosed NA. MRI of the brain and/or spinal cord was performed using high-field (1.5 T) or low-field (0.25 T) scanners using various combinations of transverse, sagittal, dorsal and three-dimensional (3D) T1-weighted (T1W), transverse, sagittal and dorsal T2-weighted (T2W), T2W fluid-attenuated inversion recovery (FLAIR) and T2*-weighted (T2*W) gradient echo (GRE), dorsal T2W short tau inversion recovery (STIR) and post-gadolinium transverse, sagittal, dorsal and 3D T1W and transverse T2W FLAIR sequences. In 4/6 cases where the brain was imaged, changes consistent with diffuse meningoencephalitis were observed. Evidence of meningeal involvement was evident even when not clinically apparent. The spinal cord was imaged in 9 dogs, with evidence of meningitis and myelitis detected in regions consistent with the observed neuroanatomical localization. Pathognomonic changes of neural larva migrans, as described in some human patients with NA, were not detected. NA should be considered in the differential diagnosis of dogs with MRI evidence of focal or diffuse meningitis, myelitis and/or encephalitis, especially in areas where A. cantonensis is endemic. If not precluded by imaging findings suggestive of brain herniation, cerebrospinal fluid (CSF) collection for cytology, fluid analysis, real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) testing should be considered mandatory in such cases after the MRI studies.

Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010-2020) and results for a novel, highly sensitive qPCR assay.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells µL-1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.

The mitochondrial genome of Angiostrongylus mackerrasae is distinct from A. cantonensis and A. malaysiensis.

The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

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.

Accurate identification of Australian mosquitoes using protein profiling.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

Occurrence, diagnosis and follow-up of canine strongyloidiosis in naturally infected shelter dogs.

Strongyloidiosis by Strongyloides stercoralis is a disease of increasing interest in human and animal medicine. The scientific knowledge on canine strongyloidiosis is hindered by the poor diagnostics available. To assess the most sensitive and specific diagnostic method, feces and blood from 100 shelter dogs were screened for S. stercoralis by coprological, molecular and serological tests. Thirty-six dogs (36%) scored positive to S. stercoralis by coprology (22.3% to Baermann) and/or 30% to real time-polymerase chain reaction (rt-PCR). According to two composite reference standards (CRS) based on all coprological methods and rt-PCR (first CRS) or in combination with serology (second CRS), the most sensitive test was IFAT (93.8%; CI 82.8-98.7), followed by rt-PCR (80.6%; 95% CI 64-91.8) and Baermann (60.6%; 95% CI 42.1-77.1). The inconsistent shedding of L1 during the 4-week follow-up in infected dogs suggests the importance of multiple faecal collections for a reliable diagnosis. A combination of serological and coprological tests is recommended for the surveillance and diagnosis of S. stercoralis infection in dogs.

Pathology Associated With an Outbreak of Entamoebiasis in Wild Cane Toads (Rhinella marina) in Tropical Australia.

Infection due to Entamoeba spp. is known to cause serious disease in primates (Entamoeba histolytica) and snakes (Entamoeba invadens), but there are no detailed descriptions of the pathology associated with Entamoeba spp. infection in amphibians. In 2014, an outbreak of entamoebiasis associated with a novel species of Entamoeba induced clinical illness and poor body condition in free-ranging cane toads in Australia's Northern Territory. Here, we describe the gross pathology, histology, and clinical pathology linked to the outbreak. The study compared 25 toads with invasive entamoebiasis, defined as histologically visible amoebas within tissue, and 12 toads without invasive entamoebiasis. Grossly, affected toads had mild to marked congestion of colonic serosal vasculature, with variable thickening of the intestinal wall and serosanguineous to hemorrhagic colonic content. Histologically, invasive entamoebiasis manifested primarily as moderate to severe, variably hyperplastic to ulcerative colitis. The small intestine was affected in 10 of 25 toads, and 5 of 25 toads also had gastric lesions. Amoebas consistent in morphology with Entamoeba sp. were commonly intermingled with mucosal epithelium, frequently along the basement membrane, with deeper invasion into the superficial lamina propria in only 5 toads. Toads with invasive entamoebiasis had neutrophilia, monocytosis, and lymphopenia, and thus elevated neutrophil to lymphocyte ratios, suggestive of an inflammatory and/or stress leukogram.

Invasive Colonic Entamoebiasis in Wild Cane Toads, Australia.

We detected a disease syndrome in free-ranging Australian cane toads involving atypical behavior and emaciation that is associated with a previously undescribed Entamoeba sp. that infiltrates the colonic lining, causing it to slough. The organism may become seasonally pathogenic when toads are under hydric and nutritional stress.

DNA barcoding of Cryptosporidium.

Cryptosporidium spp. (Apicomplexa) causing cryptosporidiosis are of medical and veterinary significance. The genus Cryptosporidium has benefited from the application of what is considered a DNA-barcoding approach, even before the term 'DNA barcoding' was formally coined. Here, the objective to define the DNA barcode diversity of Cryptosporidium infecting mammals is reviewed and considered to be accomplished. Within the Cryptosporidium literature, the distinction between DNA barcoding and DNA taxonomy is indistinct. DNA barcoding and DNA taxonomy are examined using the latest additions to the growing spectrum of named Cryptosporidium species and within-species and between-species identity is revisited. Ease and availability of whole-genome DNA sequencing of the relatively small Cryptosporidium genome offer an initial perspective on the intra-host diversity. The opportunity emerges to apply a metagenomic approach to purified field/clinical Cryptosporidum isolates. The outstanding question remains a reliable definition of Cryptosporidium phenotype. The complementary experimental infections and metagenome approach will need to be applied simultaneously to address Cryptosporidium phenotype with carefully chosen clinical evaluations enabling identification of virulence factors.

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.