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Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Editorial Board of Veterinary Parasitology (JŠ)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Editorial Board of Veterinary Parasitology (JS)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ctenocephalides felis, the cat flea, is among the most prevalent and widely dispersed vectors worldwide. Unfortunately, research on C. felis and associated pathogens (Bartonella and Rickettsia spp.) lags behind that of other vectors and vector-borne pathogens. Therefore, we aimed to review fundamental aspects of C. felis as a vector (behavior, epidemiology, phylogenetics, immunology, and microbiome composition) with an emphasis on key techniques and research avenues employed in other vector species. Future laboratory C. felis experimental infections with Bartonella, Rickettsia, and Wolbachia species/strains should examine the vector-pathogen interface utilizing contemporary visualization, transcriptomic, and gene-editing techniques. Further environmental sampling will inform the range and prevalence of C. felis and associated pathogens, improving the accuracy of vector and pathogen modeling to improve infection/infestation risk assessment and diagnostic recommendations., Competing Interests: Declaration of interests In conjunction with Sushama Sontakke and North Carolina State University, E.B.B. DVM holds US Patent No. 7,115,385: ‘Media and Methods for Cultivation of Microorganisms’, issued 3 October 2006. He is a co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella species infections as well as a scientific consultant to IDEXX Laboratories (Westbrook, ME, USA). All other authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., (© 2024. The Author(s).)

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott's test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott's test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R 2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott's test for quantitative enumeration of microfilariae (Cohen's kappa coefficient [κ]: κ = 1 using D. immitis qPCR marker, κ = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott's test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Editorial Board of Veterinary Parasitology (JS)., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within β-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Giardia duodenalis is the causative agent of the neglected diarrhoeal disease giardiasis. While often self-limiting, giardiasis is ubiquitous and impacts hundreds of millions of people annually. It is also a common gastro-intestinal disease of domestic pets, wildlife, and livestock animals. However, despite this impact, there is no vaccine for Giardia currently available. In addition, treatment relies on chemotherapies that are associated with increasing failure rates. To identify new treatment options for giardiasis we recently screened the Compounds Australia Scaffold Library for new chemotypes with selective anti-Giardia activity, identifying three compounds with sub-μM activity and promising selectivity. Here we extended these studies by examining the anti-Giardia activity of series CL9569 compounds. This compound series was of interest given the promising activity (IC 50 1.2 μM) and selectivity demonstrated by representative compound, SN00798525 (1). Data from this work has identified an additional three thieno [3,2-b]pyrrole 5-carboxamides with anti-Giardia activity, including 2 which displayed potent cytocidal (IC 50  ≤ 10 nM) and selective activity against multiple Giardia strains, including representatives from both human-infecting assemblages and metronidazole resistant parasites. Preclinical studies in mice also demonstrated that 2 is well-tolerated, does not impact the normal gut microbiota and can reduce Giardia parasite burden in these animals., Competing Interests: Declarations of competing interest CJS Hart, AG Riches, JH Ryan, and TS Skinner-Adams, are inventors on a PCT application that covers small molecule compounds derived from the compounds discussed in this manuscript., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neural angiostrongyliasis (NA) is a parasitic disease caused by Angiostrongylus cantonensis (rat lungworm). This study presents a case of NA in a captive Bolivian squirrel monkey from a zoo in western Sydney, Australia. The objective was to identify the A. cantonensis cox 1 haplotype responsible for the infection and compare its mitochondrial DNA (mtDNA) to known Australian mtDNA. An epidemiological investigation was conducted to assess the risk of infection, focusing on the resident rat population in the zoo. Methods involved trapping rats and collecting rat faeces for Angiostrongylus detection, speciation, and cox 1 haplotype confirmation. Various techniques were employed, including necropsy, morphological examination, and molecular methods such as ITS-2 qPCR, cox 1 sequencing, and ITS-2 metabarcoding. Cluster analysis of rat faeces distribution and Angiostrongylus detection utilised an equal sampling effort (ESE) approach. Gastropods were collected throughout the study for Angiostrongylus surveillance using a hypersensitive qPCR assay. Results revealed significant clustering of rat faeces near exhibits with fresh food provision and absence of predators. Angiostrongylus -positive faeces were uniformly distributed across the zoo property. Mitochondrial DNA analysis confirmed the presence of the Ac13 haplotype of A. cantonensis in the monkey. Morphology, ITS-2 metabarcoding and partial cox 1 sequencing detected only A. cantonensis , with the Ac13 cox 1 haplotype predominating. A high prevalence of infection (64%, 9/14) was found in brown rats, with quantification of larvae indicating high shedding rates. Co-infections with both Ac13 and local SYD.1 A. cantonensis cox 1 haplotypes were observed. Only three gastropods (all of which were Angiostrongylus -negative) were found in the survey. To minimise the risk of exposure for susceptible species, targeted rodent control was implemented in areas with higher exposure risk. A potential strategy (which requires further exploration) to consider for future zoo design was suggested. This study provides insights into the epidemiology and genetic diversity of A. cantonensis in Australia, emphasising the importance of control measures to prevent future outbreaks., Competing Interests: The authors have none to declare., (© 2023 The Authors.)

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.

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 β -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 β -tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta , known from domestic goats in France. Of the gastrointestinal nematodes detected via β -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.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3 rd 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.

Gastrointestinal nematodes are the most expensive agent of disease currently facing the livestock production industry. Spending the beginning of their life cycle as eggs and free-living larvae, nematodes are vulnerable to a multitude of external environmental factors. Fire is a naturally occurring force of nature that has both destructive and reconstructive effects on soil characteristics which nematode stages rely on for survival. The aim of this project was to evaluate in vitro the effect of burned pasture soil (200 °C and 500 °C) on the free-living stages of ruminant nematodes. We tested the effect of burned soil on the ability of eggs to hatch and produce infective larvae, and then tested survival of infective larvae within burned soil. Adding burned soil (500 °C) to larval cultures improved larval yield compared to larval cultures containing raw soil or soil burned at a lower heat (200 °C), and raw soil improved longer term survival of infective larvae. We were able to recover significantly more larvae from samples with low soil content either as raw or soil burned at 200 °C, when compared with samples with soil burned at 500 °C. This study has shown that the survival of gastrointestinal nematodes at the L3 stage is negatively impacted by the addition of soil burned at 500 °C. Although this temperature is closest to that of a medium intensity wildfire, which is a typically destructive process in agriculture, it reduces the number of infective GIN larvae available for animals to ingest. These experiments enable us to address in vitro if post-fire soil conditions alter the number of infective larvae available on pasture, and thus the infectivity of the pasture to livestock., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Trichomonas vaginalis is the most prevalent, non-viral sexually transmitted human infection, causing 170 million cases of trichomoniasis annually. Since the 1950s, treatment has relied on 5-nitroimidazoles (5NIs), leading to increasing drug resistance. A similar drug resistance problem is present in the veterinary pathogen, Tritrichomonas foetus. There are currently no agreed standards for defining 5NI resistance, due in part to two distinct oxygen-dependent ("aerobic") and oxygen-independent ("anaerobic") resistance phenotypes. Diagnostic tools to detect 5NI resistance are lacking, and current assays used to phenotypically assess 5NI resistance in vitro are complicated by these two resistance phenotypes. We demonstrate that microaerophilic conditions support sufficient parasite growth to interrogate oxygen-dependent resistance of 5NIs against known resistant and susceptible isolates of T. vaginalis and T. foetus. We further demonstrate that microaerophilic conditions allow sufficient growth for compatibility with existing growth assays, including our TriTOX assay. Adopting microaerophilic conditions eliminates traditional 'by-eye' estimates of minimum inhibitory concentrations and opens up options for increased throughput and automation, scalable to higher-throughput analyses of 5NI resistance. This would further allow the development of quantitative phenotypic standards to benchmark oxygen-dependent or oxygen-independent trichomonad 5NI resistance towards standardised surveillance programs to combat drug resistance., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Large language models, such as ChatGPT, will have far-reaching impacts on parasitology, including on students. Authentic experiences gained during students' training are absent from these models. This is not a weakness of the models but rather an opportunity benefiting parasitology at large., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia., Methodology/principal Findings: Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1., Conclusions/significance: The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Baláž et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. The Author(s).)

The brown dog tick Rhipicephalus sanguineus ( sensu lato ) in the southeastern Mediterranean region and the Middle East is difficult to identify due to the presence of multiple mitochondrial DNA haplogroup lineages. The purpose of this study was to clarify the identity of the "southeastern Europe" lineage of this tick species complex. Our research shows that female ticks of the "southeastern Europe" lineage correspond to the morphology of R. rutilus Koch, 1844 as found in type-material at the Museum für Naturkunde Berlin in Germany. We characterised the complete mitogenomes of R. rutilus , R. turanicus Pomerantsev, 1940 and Rhipicephalus sanguineus (Latreille, 1806) in order to improve our understanding of the phylogenetic relationships among species within the R. sanguineus ( sensu lato ) complex. The material associated with the morphology of R. rutilus was previously labelled as the "southeastern Europe" lineage and found in Israel and Egypt, including Lower Egypt and the Nile Delta, where the original type-material was collected. Based on the morphology, genetic identity, and geographical distribution of the species, we conclude that the name R. rutilus is correctly linked to the "southeastern Europe" lineage of R. sanguineus ( sensu lato )., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Published by Elsevier B.V.)

Angiostrongylus cantonensis is a nematode with an indirect lifecycle, using molluscs as intermediate hosts. Rats are the definitive host. By administering a suitable anthelmintic, at an appropriate interval, the risk of clinical neuroangiostrongyliasis occurring in paratenic hosts (e.g., dogs, man) can be eliminated. We wanted to determine if infective larvae (L3) of A. cantonensis can be safely killed during their migration through the central nervous system (CNS) by oral administration of an anthelmintic combination containing moxidectin (480 µg/kg, Simparica Trio™; M-S-P), thereby preventing patent infections in rats. Eighteen rats were used: ten received oral M-S-P every four weeks; eight rats were used as controls. Rats were initially given M-S-P as a chew to eat, but an acquired food aversion meant that subsequent doses were given by orogastric lavage. All 18 rats were challenged once or twice with approximately 30 L3 A. cantonensis larvae via orogastric lavage. Infection status was determined by faecal analysis using the Baermann technique and necropsy examination of the heart, pulmonary arteries and lungs. Eight out of ten rats dosed with M-S-P had zero lungworms at necropsy; a single female worm was detected in each of the remaining two rats. No treated rats had L1 larvae in faeces. In contrast, all eight controls were infected with patent infections, with a median of 14.5 worms per rat detected at necropsy. The difference in infection rates was significant (two tailed Fishers Exact; p = 0.0011). Moxidectin given orally once every month killed migrating larvae before they reached the pulmonary arteries in 80% of treated rats, while in 20%, only a single female worm was present. Considering the short half-life of moxidectin in the rat, it is likely that the effectiveness of moxidectin is due to larvicidal action on migrating L3, L4 and L5 larvae in the brain parenchyma or subarachnoid space, either 7 days (L3/L4 in cerebrum and spinal cord) or 14 days (L4/L5 in cerebrum and subarachnoid space) after inoculation. This study is a prelude for future research to determine if monthly moxidectin administration orally as M-S-P could prevent symptomatic neuroangiostrongyliasis in dogs.

Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic approaches to examine the sensitivity to ML drugs in D. immitis in Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knott's test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotype observed by utilising sequential quantification of microfilariae for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving 'rigorous' heartworm prevention for 12 months prior to the study, according to the owners' assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knott's test can be substituted by qPCR targeting D. immitis or associated Wolbachia endosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We re-describe the adult stages of Rhipicephalus linnaei (Audouin, 1826), and characterise its diagnostic molecular traits. A male R. linnaei collected in Esna City, Luxor Governorate, Egypt is designated as the neotype. Rhipicephalus linnaei is re-established as a valid tick name and removed from the synonymy list of Rhipicephalus sanguineus (Latreille, 1806). Rhipicephalus linnaei is most similar to R. sanguineus and Rhipicephalus camicasi Morel, Mouchet & Rodhain, 1976 because they share similar elongated comma-like spiracula that are narrowly visible dorsally, and the dorsal prolongation is narrower than the width of the adjacent festoon. The male of R. camicasi is distinguished from R. linnaei by the non-tapering caudal widening of the spiracula. The male of R. sanguineus is distinguished from R. linnaei by shorter extension that does not taper into a long narrow extension of the spiracula. The genital pore atrium of female R. linnaei is broadly U-shaped, while it is a narrower U-shape in R. sanguineus. The remaining species within the R. sanguineus species complex - Rhipicephalus sulcatus Neumann, 1908, Rhipicephalus turanicus Pomerantsev, 1940, Rhipicephalus guilhoni Morel & Vassilades, 1963, Rhipicephalus secundus Feldman-Muhsam, 1952 and Rhipicephalus afranicus Bakkes, 2020, all exhibit spiracula with the dorsal prolongation as wide as the adjacent festoon. The DNA sequence of R. linnaei is most closely related to R. guilhoni. The phylogenetic analysis of mitogenome (mtDNA) sequences including assembled mtDNA from whole genome sequencing of the neotype supports R. linnaei as a well-defined taxon when compared with DNA sequences of other species of the R. sanguineus species complex, in particular: R. sanguineus, R. turanicus, R. secundus and R. camicasi. Molecularly, R. linnaei belongs to the so-called R. sanguineus s.l. "tropical lineage" distributed globally including the Americas, Africa, Europe, Asia and is the only species from R. sanguineus species complex in Australia., (Copyright © 2022. Published by Elsevier GmbH.)

Background: The Silk Road connected the East and West for over 1500 years. Countries in Central Asia are valuable in addressing the hypothesis that parasites on domestic animals were introduced along the Silk Road. Adult fleas are obligate parasites, having worldwide distribution. In dogs, Ctenocephalides canis, C. felis and C. orientis are the most common species identified. The distribution of the Oriental cat flea, C. orientis, is restricted to southeast Asia. The purpose of this study was to determine the diversity of dog fleas from Uzbekistan, a country in Central Asia, with particular reference to C. orientis., Methods: Fleas were collected from 77 dogs from 5 locations in Uzbekistan. The cox1 gene sequences from Ctenocephalides spp. were compared to global collection of Ctenocephalides cox1 haplotypes. Landmark-based geometric morphometrics have been applied to the head and curvature to compare C. canis and C. canis using canonical variate analysis and discriminant function analysis., Results: Overall, 199 fleas were collected and identified as C. canis (n = 115, 58%), C. orientis (n = 53, 27%) and Pulex irritans (n = 22, 11%). None of the fleas were C. felis. All Ctenocephalides spp. fleas were subject to cox1 amplification and 95% (166/175) yielded DNA sequence. There were 25 cox1 haplotypes; 14 (22/25, 88%) were C. canis cox1 haplotypes and 3 (3/25, 12%) were C. orientis cox1 haplotypes. Molecular analysis confirmed the absence of C. felis. Four (4/22) and one (1/3) cox1 haplotypes were identical to cox1 haplotypes belonging to C. canis and C. orientis cox1 haplotypes identified elsewhere, respectively. Overall morphometric analysis confirmed significant differences between the head shape of C. canis and C. orientis and improved four-fivefold the species identification compared to traditional morphological key., Conclusion: We report for the first time the presence of C. orientis in Uzbekistan. Differentiation of C. orientis from C. canis and C. felis remains difficult in regions where these species coexist. Studies in Central and Southeast Asia should confirm species identity using cox1 locus to enable retracing of the distribution of the Ctenocephalides in Asia. The presence of C. orientis suggests that this species may have been introduced from the east along the ancient Silk Road., (© 2022. The Author(s).)

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 2019–2020. 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 predator–prey 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.

Background: Among protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers., Methods: A species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis., Results: A total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97-100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties., Conclusion: These findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established. GRAPHICAL ABTRACT., (© 2022. The Author(s).)

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional 'gold-standard' approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAK G2 , with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAK G2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAK G2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAK G2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAK G2 , and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAK G2 egg nemabiome metabarcoding approach., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Giardia duodenalis is one of the most common intestinal parasites of humans, with a worldwide distribution. Giardia duodenalis has been reported in both wild and captive populations of non-human primates, namely chimpanzees. In this study we investigated an entire troop of clinically healthy chimpanzees (n = 21) for the presence of G. duodenalis and its association with faecal microbiota profile. Faecal samples (n = 26) were collected from the chimpanzee exhibit from a zoo in Sydney, Australia. Diagnosis of G. duodenalis was made using a Rapid Antigen Test (RAT) as a point-of-care-test and compared to a reference standard real-time PCR test. Approximately half of the chimpanzee faecal samples tested positive for G. duodenalis by both RAT (13/26, 50%) and real-time PCR (14/26, 53.85%). The RAT sensitivity was 85.7% (95% CI: 63.8%-96%) and specificity was 91.7% (95% CI: 68.3%-99%) when compared to the in-house real-time PCR. Genotyping of the samples revealed the presence of zoonotic assemblage B. Microscopic analysis revealed the presence of Troglodytella spp. (14/26), Balantioides sp. (syn. Balantidium sp.) (8/26) as well as Entamoeba spp. (3/26). Microbiota profile based on 16S rRNA gene sequencing revealed that the community was significantly different between G. duodenalis positive and negative samples if RAT results were taken into an account, but not real-time PCR diagnostics results. Proteobacteria and Chloroflexi were the significant features in the dataset that separated G. duodenalis positive and negative samples using LEfSe analysis. Being able to rapidly test for G. duodenalis in captive populations of primates assists in point-of-care diagnostics and may better identify animals with subclinical disease. Under the investigated conditions of the zoo setting, however, presence of G. duodenalis either detected by RAT or real-time PCR was not associated with clinically apparent disease in captive chimpanzees., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tritrichomonas foetus causes chronic large-bowel diarrhoea in cats, complicated not only by its chronicity but for its difficulty to treat, with few registered or effective products available, subsequently resulting in ronidazole frequently used off-label. The extent of T. foetus resistance to ronidazole is unknown. To demonstrate in vitro resistance of feline T. foetus to ronidazole and the self-resolution of the infection, we isolated novel T. foetus "feline" genotype strains from two cats in 2017 from the same household and monitored these cats over a three-year period. Axenic cultures of T. foetus from the cats were tested for in vitro resistance to metronidazole and ronidazole using anaerobic and aerobic culture assays. The minimum lethal concentration for the novel strains after incubation in aerobic conditions for 48 h were 50-100 μg/ml and 6.25-12.50 μg/ml for metronidazole and ronidazole, respectively. For susceptible T. foetus strains, the minimum lethal concentrations ranged from 1.56-12.50 μg/ml for metronidazole, and 0.39-3.13 μg/ml for ronidazole. The self-resolution of the infection was assessed using repeat diagnostic qPCR and culture. Whilst positive in 2018, the cat samples are no longer returning positive qPCR results in 2020 and 2021, indicating self-resolution over this period. This study demonstrated resistance to ronidazole for the first time in Australian cats and infection self-resolution without antimicrobial intervention is demonstrated. This study provides clinicians with the evidence to reduce the use of off-label ronidazole, and advice on the resolution timeframe for cats in single households., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Whether subclinical shedding of canine parvovirus (CPV) by cats might contribute to the epidemiology of canine CPV infections, particularly in facilities housing both cats and dogs, requires clarification. Conflicting results are reported to date. Using conventional PCR (cPCR) to amplify the VP2 gene, shedding of the CPV variants (CPV-2a, 2b, 2c) by healthy cats in multi-cat environments was reportedly common in Europe but rare in Australia. The aim of this study was to determine whether low-level faecal CPV shedding occurs in multi-cat environments in Australia and Italy using a TaqMan real-time PCR to detect Carnivore protoparvovirus 1 (CPV and feline parvovirus, FPV) DNA, and minor-groove binder probe real-time PCR assay to differentiate FPV and CPV types and to characterize CPV variants. In total, 741 non-diarrhoeic faecal samples from shelters in Australia (n = 263) and from shelters or cat colonies in Italy (n = 478) were tested. Overall, Carnivore protoparvovirus 1 DNA was detected in 49 of 741 (6.61 %) samples. Differentiation was possible for 31 positive samples. FPV was most common among positive samples (28/31, 90.3 %). CPV was detected in 4/31 samples (12.9 %) including CPV-2a in one sample, CPV-2b in another and co-infections of FPV/CPV-2b and CPV-2a/CPV-2b in the remaining two samples. A high rate of subclinical FPV infection was detected in one shelter during an outbreak of feline panleukopenia, during which 21 of 22 asymptomatic cats (95.5 %) sampled were shedding FPV. Faecal shedding of CPV by cats in multi-cat environments is uncommon suggesting that domestic cats are not significant reservoirs of CPV., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s).)

The cat flea ( Ctenocephalides felis ) is the most common flea species parasitising both domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as vector-borne Rickettsia spp. and Bartonella spp. and could theoretically transmit Coxiella burnetii , the causative agent of Q fever. A total of 107 fleas were collected from 21 cats and 14 dogs in veterinary clinics, a feline rescue organisation and a grooming salon in New South Wales, Australia, to undergo PCR detection of Bartonella spp., Rickettsia spp. and C. burnetii DNA. Morphological identification confirmed that the cat flea ( C. felis ) is the most common flea in New South Wales, Australia, with only a single stick fast flea, Echidnophaga gallinacea recorded. The examined fleas ( n  = 35) at the cox 1 locus revealed five closely related C. felis haplotypes (inter-haplotype distance < 0.5%). Multiplex TaqMan qPCR targeting the gltA ( Rickettsia spp.) and ssrA ( Bartonella spp.) genes was positive in 22.9% (95% CI: 11.8-39.3%) and 11.4% (95% CI: 3.9-26.6%) of samples, respectively. None of the DNA isolated from fleas was positive on TaqMan qPCRs targeting the C. burnetii IS 1111 , Com 1 and htp AB genes. Co-infection of C. felis with Bartonella henselae and Bartonella clarridgeiae was demonstrated using gltA and ssrA Illumina next-generation amplicon sequencing. These findings reinforce the importance of flea control on domestic dogs and cats to effectively control the transmission of Rickettsia felis and Bartonella spp. The flea, however, is unlikely to be a vector of C. burnetii between companion animals and humans., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella . Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts. Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections. Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections. Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing. Results. In total, we identified 74 Ctenocephalides felis , two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella , all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae , Bartonella clarridgeiae and Bartonella koehlerae . Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples. Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

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 ( cox 1) 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 cox 1 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 cox 1 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 cox 1 haplotypes., (© 2021 The Author(s).)

Conservation efforts have led to the recovery of the endangered mountain gorilla populations. Due to their limited potential for spatial expansion, population densities increased, which may alter the epidemiology of infectious diseases. Recently, clinical gastrointestinal illnesses linked to helminth infections have been recorded in both gorilla populations. To understand drivers and patterns of helminth infections we quantified strongylid and tapeworm infections across both Virunga Massif and Bwindi populations using fecal egg counts. We assessed the impact of age, sex, group size, season and spatial differences used as a proxy, which reflects observed variation in the occurrence of gastrointestinal problems, vegetation types, gorilla subpopulation growth and associated social structure on helminth infections. We revealed striking geographic differences in strongylid infections with higher egg counts mostly in areas with high occurrences of gastrointestinal disease. Increased helminth egg counts were also associated with decreasing group size in some areas. Observed spatial differences may reflect mutual effects of variations in subpopulation growth rates, gorilla social structure, and vegetation associated with altitude across mountain gorilla habitat. Helminth infection intensities in Virunga gorillas were lowest in the youngest and the oldest animals. Elucidating parasite infection patterns of endangered species with low genetic diversity is crucial for their conservation management.

Due to their vector capacity, ticks are ectoparasites of medical and veterinary significance. Modern sequencing tools have facilitated tick-associated microbiota studies, but these have largely focused on bacterial pathogens and symbionts. By combining 16S rRNA gene sequencing with total RNA-sequencing methods, we aimed to determine the complete microbiome and virome of questing, female Ixodes holocyclus recovered from coastal, north-eastern New South Wales (NSW), Australia. We present, for the first time, a robust and unbiased method for the identification of novel microbes in ticks that enabled us to identify bacteria, viruses, fungi and eukaryotic pathogens. The dominant bacterial endosymbionts were Candidatus Midichloria sp. Ixholo1 and Candidatus Midichloria sp. Ixholo2. Candidatus Neoehrlichia australis and Candidatus Neoehrlichia arcana were also recovered, confirming that these bacteria encompass I. holocyclus ' core microbiota. In addition, seven virus species were detected-four previously identified in I. holocyclus and three novel species. Notably, one of the four previously identified virus species has pathogenic potential based on its phylogenetic relationship to other tick-associated pathogens. No known pathogenic eukaryotes or fungi were identified. This study has revealed the microbiome and virome of female I. holocyclus from the environment in north-eastern NSW. We propose that future tick microbiome and virome studies utilize equivalent methods to provide an improved representation of the microbial diversity in ticks globally., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chandra, Harvey, Emery, Holmes and Šlapeta.)

The brown dog tick (Rhipicephalus sanguineus) parasitises dogs. Over the past decade, two distinct lineages have been recognised - R. sanguineus sensu lato "temperate lineage" and R. sanguineus sensu lato "tropical lineage". The nominal taxon R. sanguineus (Latreille, 1806) was recently associated with the "temperate lineage". We here identify the "tropical lineage" as Rhipicephalus linnaei (Audouin, 1826) using material from Australia, where no other Rhipicephalus species parasitises dogs. Whole genome sequencing of R. linnaei from Australia, Fiji and Laos, and assembly of their complete mitochondrial DNA (~15 kb) confirms the genetic identity and distinctness from all other known species within the brown dog tick species complex. Designation of the species R. linnaei is unequivocally supported by material available through the Australian National Insect Collection, Australia. Accordingly, we are formally justified in using R. linnaei for the "tropical lineage"., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors confirm that there are no known conflicts of interest associated with this publication and that there has been no significant financial support for this work that could have influenced its outcome., (© 2021 The Author(s).)

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 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.

For the past 30 years, chemoprophylaxis with macrocyclic lactone (ML) anthelmintics has been the primary strategy for canine heartworm ( Dirofilaria immitis ) control in both the USA and Australia. ML-resistant D. immitis isolates have been confirmed to exist in the USA and studies have shown that 42 single nucleotide polymorphisms (SNPs) are associated with phenotypic ML-resistance. Currently, ML-resistance has not been reported in any Australian clinical cases of canine heartworm. The aim of the study is to determine whether the 42 SNPs associated with resistance to MLs in the isolates from the USA are present in adult heartworms from a clinical case in Australia. Five adult D. immitis obtained from a dog at post-mortem (Sydney, Australia) were sequenced using the Illumina sequencing technology. The genomic analyses revealed 6 out of the 42 SNPs associated with ML-resistance to be present in our samples, 3 out of the 6 SNPs identified were nonsynonymous SNPs but not in candidate genes for ML-resistance. ML-susceptibility profile was mixed using the 42-SNP and 10-SNP models, but the 5-SNP, 3-SNP and 2-SNP models demonstrated ML susceptibility for all five individuals. In this study, the first whole-genome reference of D. immitis from Australia establishes a new baseline for comparative studies and will be valuable for tracking ML-resistance emergence., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

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., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites., Methods: We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues (brain, liver, lung, skin, gonads) sampled from native Australian wildlife, comprising four marsupials (koala, Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird (regent honeyeater, Anthochaera phrygia) and one amphibian (eastern dwarf tree frog, Litoria fallax). Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences., Results: We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians., Conclusions: To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.

Background: The tick vector Rhipicephalus microplus which transmits Babesia spp. and rickettsial pathogens has not been reported in Kenya since 1998. More recently, the pathogenic Babesia bovis has been detected in cattle blood DNA. The status of R. microplus in Kenya remains unknown. This study employed morphological and molecular tools to characterize R. microplus originating from Kenya and assess the genetic relationships between Kenyan and other African R. microplus genotypes., Methods: Ticks were collected in south-eastern Kenya (Kwale County) from cattle and characterized to investigate the existence of R. microplus. Genetic and phylogenetic relationships between the Kenyan and other annotated R. microplus reference sequences was investigated by analysis of the cytochrome c oxidase subunit 1 (cox1) gene. To further characterize Kenyan ticks, we generated low coverage whole genome sequences of two R. microplus, one R. decoloratus and R. appendiculatus. A B. bovis specific TaqMan probe qPCR assay was used to detect B. bovis in gDNA from R. microplus ticks., Results: Occurrence of R. microplus was confirmed in Kwale County, Kenya. The Kenyan R. microplus cox1 sequences showed very high pairwise identities (> 99%) and clustered very closely with reference African R. microplus sequences. We found a low genetic variation and lack of geographical sub-structuring among the African cox1 sequences of R. microplus. Four complete mitochondrial (mt) genomes for two R. microplus, one R. decoloratus and one R. appendiculatus were assembled from next generation sequence data. The mitochondrial genome sequences of the two Kenyan R. microplus ticks clustered closely with reference genome sequences from Brazil, USA, Cambodia and India forming R. microplus Clade A. No B. bovis was detected in the Kwale R. microplus DNA., Conclusions: These findings confirm the presence of R. microplus in Kenya and suggest that R. microplus Clade A is prevalent in cattle in sub-Saharan Africa. These and other recent findings of widespread occurrence of R. microplus in Africa provide a strong justification for urgent surveillance to determine and monitor the spread of R. microplus and vector competence of Boophilus ticks for B. bovis in Africa, with the ultimate goal of strategic control.

Cat fleas ( Ctenocephalides felis ) are the most commonly recognised ectoparasites of domestic pets globally and are frequently implicated in the transmission of a variety of zoonotic vector-borne pathogens. The aim of the present study was to investigate the morphological and molecular identity of fleas parasitising cats and dogs in Northern Laos and screen them for a range of bacterial pathogens. Fleas (n = 120) were collected from dogs and cats and morphologically identified as Ctenocephalides felis (115/120) , Ctenocephalides orientis (4/120) and Pulex irritans (1/120). Molecular barcoding using the cytochrome c oxidase subunit I gene ( cox1 ) was used to confirmed species identity of 21 selected fleas. The cat flea ( C. felis ) was the most dominant flea identified. Rickettsia and Bartonella spp. DNA was detected in 21/21 and 7/21 samples, respectively, via a multiplex real-time PCR targeting gltA and ssr A. Sequencing of the seven Bartonella -positive samples and ten Rickettsia -positive samples revealed Bartonella clarridgeiae, Bartonella rochalimae , Rickettsia felis and Rickettsia sp. genotype RF2125 DNA. Anaplasma platys DNA was detected in a single C. felis after 20 of the 21 DNA samples were screened using a commercial PCR panel for vector-borne pathogens. The detection of a range of bacterial pathogens in fleas from owned cats and dogs in Northern Laos provides further evidence to the importance of these ectoparasites as vectors of zoonotic diseases in the region., (© 2020 The Author(s).)

Ticks are haematophagous ectoparasites of medical and veterinary significance due to their excellent vector capacity. Modern sequencing techniques enabled the rapid sequencing of bacterial pathogens and symbionts. This study's aims were two-fold; to determine the nymph diversity in Sydney, and to determine whether external biotic factors affect the microbiota. Tick DNA was isolated, and the molecular identity was determined for nymphs at the cox 1 level. The tick DNA was subjected to high throughput DNA sequencing to determine the bacterial profile and the impact of biotic factors on the microbiota. Four nymph tick species were recovered from Sydney, NSW: Haemaphysalis bancrofti , Ixodes holocyclus , Ixodes trichosuri and Ixodes tasmani . Biotic factors, notably tick species and geography, were found to have a significance influence on the microbiota. The microbial analyses revealed that Sydney ticks display a core microbiota. The dominating endosymbionts among all tick species were Candidatus Midichloria sp. Ixholo1 and Candidatus Midichloria sp. Ixholo2. A novel Candidatus Midichloria sp. OTU&#95;2090 was only found in I. holocyclus ticks (nymph: 96.3%, adult: 75.6%). Candidatus Neoehrlichia australis and Candidatus Neoehrlichia arcana was recovered from I. holocyclus and one I. trichosuri nymph ticks. Borrelia spp. was absent from all ticks. This study has shown that nymph and adult ticks carry different bacteria, and a tick bite in Sydney, Australia will result in different bacterial transfer depending on tick life stage, tick species and geography.

The recent detection of a novel amoebozoan parasite ( Entamoeba sp. CT1) killing invasive cane toads ( Rhinella marina) in tropical Australia raises concerns of potential spill-over into native anuran populations. Considering the vulnerability of anuran communities globally, Entamoeba sp. CT1 may pose a serious threat to anuran biodiversity. Through PCR-based detection and molecular identification, we investigated the prevalence of Entamoeba spp. in the faeces and colon tissue of cane toads ( Rhinella marina ) and eleven native Australian frog species from a single locality in the Northern Territory. No Entamoeba DNA was detected in samples of native frog faeces (N = 57) or colons (N = 17). Entamoeba DNA was detected in 24% of 45 cane toads (95%CI 14.08-38.82). Both E. ranarum and Entamoeba sp. CT1 were present in cane toads. The failure of faecal samples to indicate Entamoeba spp. in infected cane toads may be due to cysts in faeces being shed intermittently, degraded before analysis, or impervious to lysis prior to DNA isolation. Our results suggest that native frogs do not carry the pathogen in an area where 20-30% of cane toads are infected with Entamoeba sp. CT1. We demonstrate the importance of recognising PCR inhibition prior to molecular diagnostics, and the apparent inadequacy of faecal samples for the detection of Entamoeba spp. in anurans., Competing Interests: Authors declare no conflict of interest., (© 2020 The Authors.)