Molecular analysis of human tick-bitten skin yields signatures associated with distinct spatial and temporal trajectories - A proof-of-concept study.

Tick-associated diseases present challenges due to tridirectional interactions among host-specific responses, tick toxins and salivary proteins as well as microbes. We aimed to uncover molecular mechanisms in tick-bitten skin samples (cases) and contralateral skin samples (controls) collected simultaneously from the same participants, using spatial transcriptomics. Cases and controls analysed using NanoString GeoMx Digital Spatial Profiler identified 274 upregulated and 840 downregulated differentially expressed genes (DEGs), revealing perturbations in keratinization and immune system regulation. Samples of skin biopsies taken within 72 h post tick-bite DEGs had changes in protein metabolism and viral infection pathways as compared to samples taken 3 months post tick-bite, which instead displayed significant perturbations in several epigenetic regulatory pathways, highlighting the temporal nature of the host response following tick bites. Within-individual signatures distinguished tick-bitten samples from controls and identified between-individual signatures, offering promise for future biomarker discovery to guide prognosis and therapy.

Perception of Ticks and Tick-Borne Diseases Worldwide.

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges.

Distribution and Prevalence of Theileria orientalis Genotypes in Adult Lactating Dairy Cows in South West Region of Western Australia.

Bovine anaemia caused by Theileria orientalis group (BATOG) causes significant production and economic losses in Australia's cattle industry. The pathogenic T. orientalis genotypes reported in Australian cattle are type 1 (Chitose) and type 2 (Ikeda). The present study aimed to determine the prevalence and distribution of T. orientalis genotypes in adult lactating cows in Western Australia (WA) dairy herds. A total of 100 whole blood samples from lactating cows from 10 farms were obtained and screened for T. orientalis using polymerase chain reaction (PCR). Sanger sequencing was subsequently used to characterise T. orientalis genotypes isolated from positive samples. A total of thirteen cows (13%; 95% CI: 7.1-21.2%) were positive for T. orientalis, and six out of ten farms (60%; 95% CI: 26.2-87.8%) housed at least one T. orientalis-positive cow. The distribution of T. orientalis was found to be wide and dense in the South west region of WA and the southern coast of WA. The predominant T. orientalis genotype identified was Ikeda (n = 11, 11%; 95% CI: 5.6-18.8%), while the Buffeli genotype was identified in WA for the first time, albeit at a low prevalence (n = 1, 1%; 95% CI: 0.0-5.4%). This study has provided useful epidemiological evidence on the prevalence and distribution of T. orientalis in adult lactating dairy cows in WA dairy farms, and on the importance of conducting widespread surveillance programs for the understanding of BATOG in WA.

Dermatological aspects of ticks in Australia: An update.

Ticks are ectoparasites that cause dermatologic reactions directly by their bites and indirectly as vectors of bacterial, protozoal and viral diseases. Consequences vary from minor local reactions to significant systemic sequelae and are therefore of clinical relevance to dermatologists. In this article, Australian ticks of medical importance are reviewed through the lens of dermatology.

An invasive human commensal and a native marsupial maintain tick populations at the urban fringe.

Ticks (Acari: Ixodidae) are major disease vectors globally making it increasingly important to understand how altered vertebrate communities in urban areas shape tick population dynamics. In urban landscapes of Australia, little is known about which native and introduced small mammals maintain tick populations preventing host-targeted tick management and leading to human-wildlife conflict. Here, we determined (1) larval, nymphal, and adult tick burdens on host species and potential drivers, (2) the number of ticks supported by the different host populations, and (3) the proportion of medically significant tick species feeding on the different host species in Northern Sydney. We counted 3551 ticks on 241 mammals at 15 sites and found that long-nosed bandicoots (Perameles nasuta) hosted more ticks of all life stages than other small mammals but introduced black rats (Rattus rattus) were more abundant at most sites (33%-100%) and therefore important in supporting larval and nymphal ticks in our study areas. Black rats and bandicoots hosted a greater proportion of medically significant tick species including Ixodes holocyclus than other hosts. Our results show that an introduced human commensal contributes to maintaining urban tick populations and suggests ticks could be managed by controlling rat populations on urban fringes.

A systems biology approach to better understand human tick-borne diseases.

Tick-borne diseases (TBDs) are a growing global health concern. Despite extensive studies, ill-defined tick-associated pathologies remain with unknown aetiologies. Human immunological responses after tick bite, and inter-individual variations of immune-response phenotypes, are not well characterised. Current reductive experimental methodologies limit our understanding of more complex tick-associated illness, which results from the interactions between the host, tick, and microbes. An unbiased, systems-level integration of clinical metadata and biological host data - obtained via transcriptomics, proteomics, and metabolomics - offers to drive the data-informed generation of testable hypotheses in TBDs. Advanced computational tools have rendered meaningful analysis of such large data sets feasible. This review highlights the advantages of integrative system biology approaches as essential for understanding the complex pathobiology of TBDs.

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia.

In Australia, there is a paucity of data about the extent and impact of zoonotic tick-related illnesses. Even less is understood about a multifaceted illness referred to as Debilitating Symptom Complexes Attributed to Ticks (DSCATT). Here, we describe a research plan for investigating the aetiology, pathophysiology, and clinical outcomes of human tick-associated disease in Australia. Our approach focuses on the transmission of potential pathogens and the immunological responses of the patient after a tick bite. The protocol is strengthened by prospective data collection, the recruitment of two external matched control groups, and sophisticated integrative data analysis which, collectively, will allow the robust demonstration of associations between a tick bite and the development of clinical and pathological abnormalities. Various laboratory analyses are performed including metagenomics to investigate the potential transmission of bacteria, protozoa and/or viruses during tick bite. In addition, multi-omics technology is applied to investigate links between host immune responses and potential infectious and non-infectious disease causations. Psychometric profiling is also used to investigate whether psychological attributes influence symptom development. This research will fill important knowledge gaps about tick-borne diseases. Ultimately, we hope the results will promote improved diagnostic outcomes, and inform the safe management and treatment of patients bitten by ticks in Australia.

An argument for pandemic risk management using a multidisciplinary One Health approach to governance: an Australian case study.

The emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic has resulted in significant global impact. However, COVID-19 is just one of several high-impact infectious diseases that emerged from wildlife and are linked to the human relationship with nature. The rate of emergence of new zoonoses (diseases of animal origin) is increasing, driven by human-induced environmental changes that threaten biodiversity on a global scale. This increase is directly linked to environmental drivers including biodiversity loss, climate change and unsustainable resource extraction. Australia is a biodiversity hotspot and is subject to sustained and significant environmental change, increasing the risk of it being a location for pandemic origin. Moreover, the global integration of markets means that consumption trends in Australia contributes to the risk of disease spill-over in our regional neighbours in Asia-Pacific, and beyond. Despite the clear causal link between anthropogenic pressures on the environment and increasing pandemic risks, Australia's response to the COVID-19 pandemic, like most of the world, has centred largely on public health strategies, with a clear focus on reactive management. Yet, the span of expertise and evidence relevant to the governance of pandemic risk management is much wider than public health and epidemiology. It involves animal/wildlife health, biosecurity, conservation sciences, social sciences, behavioural psychology, law, policy and economic analyses to name just a few.The authors are a team of multidisciplinary practitioners and researchers who have worked together to analyse, synthesise, and harmonise the links between pandemic risk management approaches and issues in different disciplines to provide a holistic overview of current practice, and conclude the need for reform in Australia. We discuss the adoption of a comprehensive and interdisciplinary 'One Health' approach to pandemic risk management in Australia. A key goal of the One Health approach is to be proactive in countering threats of emerging infectious diseases and zoonoses through a recognition of the interdependence between human, animal, and environmental health. Developing ways to implement a One Health approach to pandemic prevention would not only reduce the risk of future pandemics emerging in or entering Australia, but also provide a model for prevention strategies around the world.

Knowledge, Attitude and Practices Towards Cryptosporidium Among Public Swimming Pool Patrons and Staff in Western Australia.

PURPOSE: There is a dearth of research conducted on the Knowledge, Attitude and Practices (KAP) of swimming pool patrons and staff to determine their understanding of the importance of Cryptosporidium and its transmission in swimming pools. METHODS: We conducted a KAP survey of public swimming pool patrons (n = 380) and staff (n = 40) attending five public swimming pools in Western Australia (WA). RESULTS: Knowledge, attitudes and practices (KAP) of Cryptosporidium varied between patrons and staff but were generally limited. Only 26.1% and 25.0% of patrons and staff had heard of Cryptosporidium, while 17.4% and 10.0% knew that it causes diarrhoea, respectively. Thirty-one percent of patrons were aware of their pool policy concerning gastroenteritis and Cryptosporidium, compared to 62.5% of staff. Less than 50% of patrons demonstrated awareness of how features within the pool environment were relevant to the control of Cryptosporidium. Only about a third of patrons (35%) and staff (37.5%) were aware that showering before swimming reduced the risk of gastroenteritis. CONCLUSION: Raising awareness about hygiene-related practices through the delivery of targeted health education messages to the general public is essential to reduce the burden of Cryptosporidium infections in aquatic environments.

First record of the stump-tailed lizard tick, Amblyomma albolimbatum (Ixodida, Ixodidae) parasitising a human.

Ticks are haematophagous arthropods that parasitise a wide range of vertebrate hosts. In Australia, there are currently 74 tick species described; 22 tick species have been reported parasitising humans. The stump-tailed lizard tick, Amblyomma albolimbatum, feeds on reptiles, most commonly lizards and snakes; however, we report the first case of A. albolimbatum parasitising a human. The nymphal tick was removed while conducting fieldwork on western tiger snakes (Notechis scutatus occidentalis) in an urban city environment near Perth, Western Australia. The tick was identified using morphological descriptions, which was further supported by the abundance of all parasitic stages of A. albolimbatum on the tiger snakes sampled. The number of tick species recorded from humans in Australia is now revised to 23 species. With the increasing incidence of tick-borne illnesses in Australia, this study highlights the need to report cases of new or atypical hosts, particularly humans, and especially when the ticks have been associated with zoonotic pathogens.

The bacterial biome of ticks and their wildlife hosts at the urban-wildland interface.

Advances in sequencing technologies have revealed the complex and diverse microbial communities present in ticks (Ixodida). As obligate blood-feeding arthropods, ticks are responsible for a number of infectious diseases that can affect humans, livestock, domestic animals and wildlife. While cases of human tick-borne diseases continue to increase in the northern hemisphere, there has been relatively little recognition of zoonotic tick-borne pathogens in Australia. Over the past 5 years, studies using high-throughput sequencing technologies have shown that Australian ticks harbour unique and diverse bacterial communities. In the present study, free-ranging wildlife (n=203), representing ten mammal species, were sampled from urban and peri-urban areas in New South Wales (NSW), Queensland (QLD) and Western Australia (WA). Bacterial metabarcoding targeting the 16S rRNA locus was used to characterize the microbiomes of three sample types collected from wildlife: blood, ticks and tissue samples. Further sequence information was obtained for selected taxa of interest. Six tick species were identified from wildlife: Amblyomma triguttatum, Ixodes antechini, Ixodes australiensis, Ixodes holocyclus, Ixodes tasmani and Ixodes trichosuri. Bacterial 16S rRNA metabarcoding was performed on 536 samples and 65 controls, generating over 100 million sequences. Alpha diversity was significantly different between the three sample types, with tissue samples displaying the highest alpha diversity (P<0.001). Proteobacteria was the most abundant taxon identified across all sample types (37.3 %). Beta diversity analysis and ordination revealed little overlap between the three sample types (P<0.001). Taxa of interest included Anaplasmataceae, Bartonella, Borrelia, Coxiellaceae, Francisella, Midichloria, Mycoplasma and Rickettsia. Anaplasmataceae bacteria were detected in 17.7% (95/536) of samples and included Anaplasma, Ehrlichia and Neoehrlichia species. In samples from NSW, 'Ca. Neoehrlichia australis', 'Ca. Neoehrlichia arcana', Neoehrlichia sp. and Ehrlichia sp. were identified. A putative novel Ehrlichia sp. was identified from WA and Anaplasma platys was identified from QLD. Nine rodent tissue samples were positive for a novel Borrelia sp. that formed a phylogenetically distinct clade separate from the Lyme Borrelia and relapsing fever groups. This novel clade included recently identified rodent-associated Borrelia genotypes, which were described from Spain and North America. Bartonella was identified in 12.9% (69/536) of samples. Over half of these positive samples were obtained from black rats (Rattus rattus), and the dominant bacterial species identified were Bartonella coopersplainsensis and Bartonella queenslandensis. The results from the present study show the value of using unbiased high-throughput sequencing applied to samples collected from wildlife. In addition to understanding the sylvatic cycle of known vector-associated pathogens, surveillance work is important to ensure preparedness for potential zoonotic spillover events.

Molecular analysis of cryptosporidiosis cases in Western Australia in 2019 and 2020 supports the occurrence of two swimming pool associated outbreaks and reveals the emergence of a rare C. hominis IbA12G3 subtype.

Cryptosporidium is an important protozoan parasite and due to its resistance to chlorine is a major cause of swimming pool-associated gastroenteritis outbreaks. The present study combined contact tracing and molecular techniques to analyse cryptosporidiosis cases and outbreaks in Western Australia in 2019 and 2020. In the 2019 outbreak, subtyping at the 60 kDa glycoprotein (gp60) gene identified 89.0% (16/18) of samples were caused by the C. hominis IdA15G1 subtype. Amplicon next generation sequencing (NGS) at the gp60 locus identified five C. hominis IdA15G1 subtype samples that also had C. hominis IdA14 subtype DNA, while multi locus sequence typing (MLST) analysis on a subset (n = 14) of C. hominis samples identified three IdA15G1 samples with a 6 bp insertion at the end of the trinucleotide repeat region of the cp47 gene. In 2020, 88.0% (73/83) of samples typed were caused by the relatively rare C. hominis subtype IbA12G3. Four mixed infections were observed by NGS with three IdA15G1/ IdA14 mixtures and one C. parvum IIaA18G3R1 sample mixed with IIaA16G3R1. No genetic diversity using MLST was detected. Epidemiological and molecular data indicates that the outbreaks in 2019 and 2020 were each potentially from swimming pool point sources and a new C. hominis subtype IbA12G3 is emerging in Australia. The findings of the present study are important for understanding the introduction and transmission of rare Cryptosporidium subtypes to vulnerable populations.

Cryptosporidium abrahamseni n. sp. (Apicomplexa: Cryptosporidiiae) from red-eye tetra (Moenkhausia sanctaefilomenae).

The morphological, biological, and molecular characterisation of Cryptosporidium piscine genotype 7 from red-eye tetras (Moenkhausia sanctaefilomenae) are described, and the species name Cryptosporidium abrahamseni n. sp. is proposed. Histological analysis of intestinal tissue identified large numbers of Cryptosporidium organisms along the epithelial lining of the intestine. Sequence and phylogenetic analysis at 18S rRNA (18S) and actin loci conducted on intestinal scrapings revealed that C. abrahamseni n. sp. was genetically distinct from other Cryptosporidium species. At the 18S locus, it was most closely related to C. huwi (3.2% genetic distance) and exhibited genetic distances ranging from 5.9 to 6.5% (C. molnari) to 14.9% (C. scolpthalmi) from all other Cryptosporidium species. At the actin locus, the genetic distances were larger and C. abrahamseni n. sp. exhibited 10.3% genetic distance from C. huwi, and 17.6% (C. molnari) to 28% (C. canis) genetic distance from other Cryptosporidium spp. Phylogenetic analysis of concatenated 18S and actin sequences confirmed that C. abrahamseni n. sp. shares the closest genetic relationship with C. huwi (6.7% genetic distance), while the genetic distance between C. abrahamseni n. sp. and other Cryptosporidium spp. ranged from 12.1% (C. molnari) to 20.4% (C. canis). Based on genetic and histological data, C. abrahamseni n. sp. is validated as a separate species.

Zoonotic infection by Cryptosporidium fayeri IVgA10G1T1R1 in a Western Australian human.

In the present study, a 37-year-old immunosuppressed female in Western Australia (WA) was identified as positive for Cryptosporidium by microscopy and treated with nitazoxanide. Molecular analyses at the 18S ribosomal RNA (18S) and 60 kDa glycoprotein (gp60) loci identified C. fayeri subtype IVgA10G1T1R1, which had previously been identified in western grey kangaroos (Macropus fuliginosus) in WA. Next generation sequencing (NGS) of the gp60 locus confirmed the absence of mixed infections with other Cryptosporidium species. This is only the second report of C. fayeri in a human host highlighting the zoonotic potential of this wildlife-associated species. Routine diagnosis using molecular methods in laboratories is required to better understand the diversity and epidemiology of Cryptosporidium parasite.

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia.

Vector-borne haemoprotozoans comprise a diverse group of eukaryote single-celled organisms transmitted by haematophagous (blood-feeding) invertebrates. They can cause debilitating diseases that impact wildlife, livestock, companion animals and humans. Recent research has shown that Australian wildlife host a diverse range of haemoprotozoan species; however, to date this work has primarily been confined to a few host species or isolated populations in rural habitats. There has been little investigation into the presence of these blood parasites in wildlife inhabiting urban and peri-urban areas. In this study, blood and tissue samples and ticks were collected from wildlife in New South Wales and Western Australia. Extracted DNA samples were screened with pan-specific molecular assays to determine the presence of haemoprotozoans using amplicon metabarcoding and Sanger sequencing approaches. In addition, light microscopy was performed on blood films. Eight haemoprotozoans were identified in the present study, which included species of Babesia, Hepatozoon, Theileria and Trypanosoma. Blood samples were collected from 134 animals; 70 black rats (Rattus), 18 common brush-tailed possums (Trichosurus vulpecula), two bush rats (Rattus fuscipes), 22 chuditch (Dasyurus geoffroii), 20 long-nosed bandicoots (Perameles nasuta), one quenda (Isoodon fusciventer) and one swamp rat (Rattus lutreolus). Molecular screening of DNA extracted from blood samples identified 52.2% (95% CI: 43.8-60.5%) of individuals were positive for at least one haemoprotozoan species, with 19.4% (95% CI: 13.4-26.7%) positive for more than one species. The present study provides the first sequences of Theileria cf. peramelis from black rats and long-nosed bandicoots. Babesia lohae was identified from brush-tailed possums. Two Hepatozoon genotypes were identified from black rats and bush rats. Black rats showed the highest haemoprotozoan diversity, with five species identified. No known human pathogens that have been described in the northern hemisphere were identified in the present study, and future work is required to understand the zoonotic potential of these microbes in Australia. This work represents the first large-scale body of research using molecular tools to investigate haemoprotozoans in animals at the urban-wildland interface. Further research is needed to investigate potential consequences of infection in wildlife, particularly effects of pathogen spillover from invasive black rats to native wildlife.

Illuminating the bacterial microbiome of Australian ticks with 16S and Rickettsia-specific next-generation sequencing.

Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum" and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9-99.1%) to Rickettsia raoultii and Rickettsia gravesii. "Candidatus Rickettsia jingxinensis" was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illumina's 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases.

Blood Parasites in Endangered Wildlife-Trypanosomes Discovered During a Survey of Haemoprotozoa from the Tasmanian Devil.

The impact of emerging infectious diseases is increasingly recognised as a major threat to wildlife. Wild populations of the endangered Tasmanian devil, Sarcophilus harrisii, are experiencing devastating losses from a novel transmissible cancer, devil facial tumour disease (DFTD); however, despite the rapid decline of this species, there is currently no information on the presence of haemoprotozoan parasites. In the present study, 95 Tasmanian devil blood samples were collected from four populations in Tasmania, Australia, which underwent molecular screening to detect four major groups of haemoprotozoa: (i) trypanosomes, (ii) piroplasms, (iii) Hepatozoon, and (iv) haemosporidia. Sequence results revealed Trypanosoma infections in 32/95 individuals. Trypanosoma copemani was identified in 10 Tasmanian devils from three sites and a second Trypanosoma sp. was identified in 22 individuals that were grouped within the poorly described T. cyclops clade. A single blood sample was positive for Babesia sp., which most closely matched Babesia lohae. No other blood protozoan parasite DNA was detected. This study provides the first insight into haemoprotozoa from the Tasmanian devil and the first identification of Trypanosoma and Babesia in this carnivorous marsupial.

First glimpse into the origin and spread of the Asian longhorned tick, Haemaphysalis longicornis, in the United States.

Established populations of Asian longhorned ticks (ALT), Haemaphysalis longicornis, were first identified in the United States (US) in 2017 by sequencing the mitochondrial cytochrome c oxidase subunit I (cox1) 'barcoding' locus followed by morphological confirmation. Subsequent investigations detected ALT infestations in 12, mostly eastern, US states. To gain information on the origin and spread of US ALT, we (1) sequenced cox1 from ALT populations across 9 US states and (2) obtained cox1 sequences from potential source populations [China, Japan and Republic of Korea (ROK) as well as Australia, New Zealand and the Kingdom of Tonga (KOT)] both by sequencing and by downloading publicly available sequences in NCBI GenBank. Additionally, we conducted epidemiological investigations of properties near its initial detection locale in Hunterdon County, NJ, as well as a broader risk analysis for importation of ectoparasites into the area. In eastern Asian populations (China/Japan/ROK), we detected 35 cox1 haplotypes that neatly clustered into two clades with known bisexual versus parthenogenetic phenotypes. In Australia/New Zealand/KOT, we detected 10 cox1 haplotypes all falling within the parthenogenetic cluster. In the United States, we detected three differentially distributed cox1 haplotypes from the parthenogenetic cluster, supporting phenotypic evidence that US ALT are parthenogenetic. While none of the source populations examined had all three US cox1 haplotypes, a phylogeographic network analysis supports a northeast Asian source for the US populations. Within the United States, epidemiological investigations indicate ALT can be moved long distances by human transport of animals, such as horses and dogs, with smaller scale movements on wildlife. These results have relevant implications for efforts aimed at minimizing the spread of ALT in the United States and preventing additional exotic tick introductions.

Cryptosporidium bollandi n. sp. (Apicomplexa: Cryptosporidiiae) from angelfish (Pterophyllum scalare) and Oscar fish (Astronotus ocellatus).

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.