Comparison of Giardia duodenalis point-of-care antigen faecal tests to reference laboratory assays in non-symptomatic dogs.

Giardia duodenalis is one of the most prevalent enteric parasites of dogs. Point-of-care antigen tests (POC) are rapid and do not require additional equipment, or a specialised diagnostic laboratory. The aim of this study was to compare diagnostic tests available in veterinary practices and in a diagnostic laboratory for the detection of G. duodenalis on a cohort of group-housed dogs from New South Wales, Australia. Two different POC tests were used for the detection of G. duodenalis. Laboratory tests used were the multiplexed-tandem PCR panel (MT-PCR) that includes detection of G. duodenalis DNA, and two reference tests (an in-house TaqMan real-time PCR and a direct immunofluorescence assay, DFA). Canine faecal samples (n = 40) were tested simultaneously for the detection of G. duodenalis. Using either DFA or TaqMan real-time PCR as reference tests, 77.5% (31/40) and 82.5% (33/40) of dogs tested positive, respectively. Agreement (Kappa) between the DFA and TaqMan real-time PCR was 0.84 (95% CI 0.64 to 1.00). There was substantial G. duodenalis test outcome agreement between the two POC tests, Kappa = 0.75. Combining the two POC tests yielded 77% sensitivity and 100% specificity with DFA as reference, and for TaqMan real-time PCR it was 73% sensitivity and 100% specificity. The MT-PCR was in excellent agreement with each reference test, DFA or TaqMan real-time PCR. Due to the high specificity of both POC tests, they can be confidently used as rule-in diagnostics. Confirmatory testing that detects different biological parameters such as DNA, e.g. PCR (inc. MT-PCR), should be implemented before concluding that a dog is negative for the presence of G. duodenalis.

Efficacy and safety of Felpreva®, a spot-on formulation for cats containing emodepside, praziquantel and tigolaner against experimental infestation with the Australian paralysis tick Ixodes holocyclus.

The Australian paralysis tick Ixodes holocyclus continues to be a serious threat to companion animals along Australia's east coast. The tick produces a potent neurotoxin which causes a rapidly ascending flaccid paralysis, which if left untreated, can result in the death of the animal. There is currently only a limited number of products registered in Australia for the treatment and control of paralysis ticks in cats. Felpreva® is an effective spot-on combination containing emodepside, praziquantel and tigolaner. To investigate the therapeutic and long-term persistent efficacy of Felpreva® (2.04% w/v emodepside, 8.14% w/v praziquantel and 9.79% w/v tigolaner) against experimental infestation with I. holocyclus in cats, two studies were undertaken. Fifty cats were included in the studies on study Day -17. These cats were immunized against paralysis tick holocyclotoxin prior to the study commencing. Immunity to holocyclotoxin was confirmed with a tick carrying capacity (TCC) test conducted prior to treatment. Cats were treated once on Day 0. Group 1 cats were treated with the placebo formulation and Group 2 cats were treated with Felpreva®. Cats were infested on Days -14 (tick carrying capacity test), 0, 28, 56, 70, 84 and 91 (weeks 4, 8, 10, 12 and 13). Ticks were counted on cats 24 h, 48 h and 72 â€‹h post-treatment and infestation, except during the tick carrying capacity test when they were counted approximately 72 â€‹h post-infestation only. The 24-h and 48-h assessments were conducted without removing the ticks. The ticks were assessed, removed and discarded at the 72-h assessment time-points. Significant differences in total live tick counts at ∼24 h, ∼48 h and ∼72 â€‹h post-infestation were observed between the treatment and control group. Differences were significant (P â€‹< â€‹0.05 to â€‹< â€‹0.001) in all instances. Treatment efficacies of 98.1-100% were observed ∼72 â€‹h post-infestation through to 13 weeks (94 days) post-treatment. These results show that a single application of Felpreva® provides effective treatment and control against induced infestation with paralysis ticks for 13 weeks.

Protecting dogs and cats against the Australian paralysis tick, Ixodes holocyclus (Acari: Ixodidae): A review of the Australian acaricide registration process.

Tick control is mainly achieved through the use of effective ectoparasiticides that can be either dermally or systemically distributed in/on the host. Before any acaricide can be legally made available to veterinarians and pet owners, it must demonstrate efficacy in a series of well-designed dose confirmation studies. The data generated during these studies are then reviewed by government regulators and used for the registration of the acaricide. In Australia, the most significant tick species is the Australian paralysis tick, Ixodes holocyclus. This three-host tick produces a potent neurotoxin (holocyclotoxin) that induces a rapidly ascending flaccid paralysis that can be fatal to companion animals and larger mammals such as cattle and horses. The Australian Pesticides and Veterinary Medicines Authority (APVMA) is the national Australian regulator which sets the data requirements for the registration of acaricides. This paper reviews the requirements set by the APVMA and puts them in direct context with the biology, distribution and reported acaricide susceptibility of I. holocyclus. An overview of acaricides currently registered in Australia for the control of I. holocyclus in dogs and cats, their reported efficacy data and the conduct of I. holocyclus efficacy trials are also provided.

Anthelmintic resistance and common worm control practices in sheep farms in Flanders, Belgium.

In contrast to many other European countries, no data were available on the presence of anthelmintic resistance in gastrointestinal nematodes in sheep in Belgium. A faecal egg count reduction test was performed in 26 sheep flocks in Flanders, Northern Belgium. Results indicated widespread resistance against benzimidazoles (albendazole, fenbendazole and mebendazole), with treatment failure on all 8 farms investigated. Haemonchus contortus and Teladorsagia circumcincta were the predominant species after treatment failure. Amino acid substitutions associated with benzimidazole resistance were detected at the codon positions 167 (8%) and 200 (92%) of the isotype-1 beta tubulin gene in H. contortus, codon positions 198 (47%) and 200 (43%) in T. circumcincta and position 200 (100%) in T. colubriformis. Resistance against macrocyclic lactones (ivermectin, doramectin and moxidectin) was recorded on 7 out of 20 flocks, mainly in H. contortus and T. circumcincta. Treatment failure was also observed for closantel (in combination with mebendazole) and for monepantel, on one farm each. Trichostrongylus spp. were implicated with resistance against monepantel. A questionnaire survey on farm management and worm control measures indicated that worm control was often not sustainable. Ewes and lambs were treated frequently (on average 2.6 and 3.2 times per year), mostly without weighing. Only few sheep farmers (9%) regularly used faecal egg counts to monitor worm infections. Despite the FECRT showing otherwise, most of the farmers perceived the efficacy of anthelmintics as very good (30%) or good (54%).

Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats.

Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of Ct-values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice.

An automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study.

Detecting the genera and species of gastrointestinal (GI) nematode infections in faecal samples obtained from cattle requires the incubation of faeces ('larval culture') followed by identification of the third-stage larvae that are harvested after 10-14days. Substantial research in the development of PCR-based methods for the rapid and specific identification GI nematodes has been conducted for small ruminants, whilst only few such assays have been developed for cattle. In the present paper we describe the development of an automated, robotic PCR platform for the detection and genus and/or species-specific identification of GI nematodes from bovine faecal samples. This test was then validated using samples from different regions of three countries (Australia, Belgium and Scotland). The PCR platform was found to be highly sensitive and specific for the identification of the important GI nematodes in naturally infected cattle (both estimates >90%). The PCR platform can also estimate the percentage of genera or species present in a mixed-species infection, and was found superior to larval culture in terms of speed (1-2days versus 1-2 weeks for culture), sensitivity and specificity. The PCR was simple to use and the operator requires no knowledge or experience to identify the nematodes present, compared to larval culture where even experienced operators can make substantial errors due to considerable overlap in the published characteristics of key species.

Multiplexed-tandem PCR for the specific diagnosis of gastrointestinal nematode infections in sheep: an European validation study.

BACKGROUND: Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories. RESULTS: MT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific. CONCLUSIONS: The MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.

Semiquantitative multiplexed tandem PCR for detection and differentiation of four Theileria orientalis genotypes in cattle.

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.

A real-time PCR assay for the diagnosis of gastrointestinal nematode infections of small ruminants.

The diagnosis of gastrointestinal nematode infections in small ruminants is central to studying the biology and epidemiology of these parasites and underpins their control. Traditional methods of diagnosis are inaccurate, time-consuming and laborious. Here, we describe a step-by-step protocol for the molecular-based diagnosis of infections by real-time PCR.

The specific diagnosis of gastrointestinal nematode infections in livestock: larval culture technique, its limitations and alternative DNA-based approaches.

The specific diagnosis of gastrointestinal nematode infections in ruminants is routinely based on larval culture technique and on the morphological identification of developed third-stage larvae. However, research on the ecology and developmental requirements of different species suggests that environmental conditions (e.g., temperature and humidity) for optimal development to occur vary between the different species. Thus, employing a common culture protocol for all species will favour the development of certain species over others and can cause a biased result in particular when species proportions in a mixed infection are to be determined. Furthermore, the morphological identification of L3 larvae is complicated by a lack of distinctive, obvious features that would allow the identification of all key species. In the present paper we review in detail the potential limitations of larval culture technique and morphological identification and provide account to some modern molecular alternatives to the specific diagnosis of gastrointestinal nematode infection in ruminants.

Next-generation molecular-diagnostic tools for gastrointestinal nematodes of livestock, with an emphasis on small ruminants: a turning point?

Parasitic nematodes of livestock have major economic impact worldwide. Despite the diseases caused by these nematodes, some advances towards the development of new therapeutic agents and attempts to develop effective vaccines against some of them, there has been limited progress in the development of practical diagnostic methods. The specific and sensitive diagnosis of parasitic nematode infections of livestock underpins effective disease control, which is now particularly important given the problems associated with anthelmintic resistance in parasite populations. Traditional diagnostic methods have major limitations, in terms of sensitivity and specificity. This chapter provides an account of the significance of parasitic nematodes (order Strongylida), reviews conventional diagnostic techniques that are presently used routinely and describes advances in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infections. A particular emphasis is placed on the recent development of a robotic PCR-based platform for high-throughput diagnosis, and its significance and implications for epidemiological investigations and for use in control programmes.

Impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance - an Australian perspective.

Parasitic nematodes (roundworms) of small ruminants and other livestock have major economic impacts worldwide. Despite the impact of the diseases caused by these nematodes and the discovery of new therapeutic agents (anthelmintics), there has been relatively limited progress in the development of practical molecular tools to study the epidemiology of these nematodes. Specific diagnosis underpins parasite control, and the detection and monitoring of anthelmintic resistance in livestock parasites, presently a major concern around the world. The purpose of the present article is to provide a concise account of the biology and knowledge of the epidemiology of the gastrointestinal nematodes (order Strongylida), from an Australian perspective, and to emphasize the importance of utilizing advanced molecular tools for the specific diagnosis of nematode infections for refined investigations of parasite epidemiology and drug resistance detection in combination with conventional methods. It also gives a perspective on the possibility of harnessing genetic, genomic and bioinformatic technologies to better understand parasites and control parasitic diseases.

Comparative evaluation of two DNA isolation techniques for PCR-based diagnosis of gastrointestinal nematode infections in sheep.

The specific diagnosis of gastrointestinal parasite infections in livestock is central to their control. PCR assays have been developed for routine diagnosis and to overcome limitations of classical methods. Central to the performance of such assays is the effective isolation of the nucleic acids from samples and the elimination of components that are inhibitory to PCR. Here, we directly compared two techniques for the isolation of DNA from strongylid nematode eggs from faecal samples from sheep, and assessed their performance in relation to the sensitivity and specificity of PCR, time required for DNA isolation and ease of use. The results showed differences in the performance of the two isolation techniques, subsequently effecting the PCR results. The main differences related to the time required for DNA isolation, and the elimination of inhibitory substances from the DNA isolated by one technique but not the other.

Advances in the diagnosis of key gastrointestinal nematode infections of livestock, with an emphasis on small ruminants.

Parasitic nematodes (roundworms) of livestock have major economic impact globally. In spite of the diseases caused by these nematodes and some advances in the design of new therapeutic agents (anthelmintics) and attempts to develop vaccines against some of them, there has been limited progress in the establishment of practical diagnostic techniques. The specific and sensitive diagnosis of gastrointestinal nematode infections of livestock underpins effective disease control, which is highly relevant now that anthelmintic resistance (AR) is a major problem. Traditional diagnostic techniques have major constraints, in terms of sensitivity and specificity. The purpose of this article is to provide a brief background on gastrointestinal nematodes (Strongylida) of livestock and their control; to summarize conventional methods used for the diagnosis and discuss their constraints; to review key molecular-diagnostic methods and recent progress in the development of advanced amplification-based and sequencing technologies, and their implications for epidemiological investigations and the control of parasitic diseases.

Establishment of a robotic, high-throughput platform for the specific diagnosis of gastrointestinal nematode infections in sheep.

The accurate diagnosis of strongylid nematode infections is central to investigating their epidemiology and for parasite control. To overcome major limitations in sensitivity or specificity of traditional methods, including faecal egg count (FEC) and/or larval culture (LC), we evaluated and established a semi-automated, high throughput multiplexed-tandem PCR (MT-PCR) platform for the diagnosis of gastrointestinal strongylid nematode infections in sheep, and established its diagnostic sensitivity (100%) and specificity (87.5%) based on the testing of 100 faecal DNA samples from helminth-free sheep and 30 samples from sheep with infections confirmed by necropsy. Subsequently, the platform was employed to test 219 faecal samples from sheep with naturally acquired infections from various geographical localities within Australia and the results compared with those from conventional LC using 139 of the 219 samples. The results obtained using both MT-PCR and LC correlated significantly for most nematodes examined, but revealed that Oesophagostomum venulosum and Chabertia ovina (parasites of the large intestine) were significantly under-represented in the LC results. The results showed that Trichostrongylus spp. (87%), Teladorsagia circumcincta (80%) and Haemonchus contortus (67%) had the highest prevalences, followed by O. venulosum (51%) and C. ovina (12%). The molecular-diagnostic platform established can be used for species- or genus-specific diagnosis of patent nematode infections within 24h (compared with 7-10 days for LC), and is a sensitive and cost effective tool for routine application in research and service laboratories.

A molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep.

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.

Evaluation and application of a molecular method to assess the composition of strongylid nematode populations in sheep with naturally acquired infections.

We evaluated the performance of a PCR method for the diagnosis of naturally acquired strongylid nematode infections in sheep (n = 470; in a temperate climatic zone of south-eastern Australia), using a panel of 100 'negative control' samples from sheep known not to harbour parasitic helminths. We compared the diagnostic sensitivity (98%) and specificity (100%) of this assay against a conventional faecal flotation method and also established a system to rank the contribution of particular strongylid nematodes to the faecal egg counts (FECs) from 'mixed infections' in individual sheep. The testing of faecal samples herein revealed that Teladorsagia circumcincta (80%) and Trichostrongylus spp. (66%) were most prevalent, followed by Chabertia ovina (33%), Oesophagostomum venulosum (28%) and Haemonchus contortus (1%). For the majority of sheep in this study, T. circumcincta and Trichostrongylus spp. represented the largest proportion of strongylid eggs in faecal samples from individual sheep. This is the first large-scale prevalence survey of gastrointestinal nematodes in live sheep using a molecular tool. The ability to rapidly rank strongylid nematodes according to their contribution to mixed infections represents a major advantage over routine coprological methods. This PCR tool has the potential to replace the conventional technique of larval culture. Future efforts will focus on enhancing and adapting this molecular method for high throughput application in routine, diagnostic settings.

Haemangioma in the oesophagus of a red-eared slider (Trachemys scripta elegans).

A haemangioma developing in the wall of the oesophagus and protruding into its cavity is reported for the first time from a Red-eared Slider (Trachemys scripta elegans). As the tumour mechanically hampered swallowing, the animal was unable to eat and consequently developed a poor condition. Histopathology of the tumour revealed all characteristics of a haemangioma: the blood-filled blood-vessels having an irregular cross-section were lined with endothelial cells. Claudin-5 immunohistochemical antibodies were employed for characterising the tumour, and this examination confirmed our initial diagnosis of a haemangioma.