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Sheep and goats are commonly infected with three Cryptosporidium species, including Cryptosporidium parvum, Cryptosporidium ubiquitum, and Cryptosporidium xiaoi, which differ from each in prevalence, geographic distribution, and public health importance. While C. parvum appears to be a dominant species in small ruminants in European countries, its occurrence in most African, Asian, and American countries appear to be limited. As a result, zoonotic infections due to contact with lambs and goat kids are common in European countries, leading to frequent reports of outbreaks of cryptosporidiosis on petting farms. In contrast, C. xiaoi is the dominant species elsewhere, and mostly does not infect humans. While C. ubiquitum is another zoonotic species, it occurs in sheep and goats at much lower frequency. Host adaptation appears to be present in both C. parvum and C. ubiquitum, consisting of several subtype families with different host preference. The host-adapted nature of C. parvum and C. ubiquitum has allowed the use of subtyping tools in tracking infection sources. This has led to the identification of geographic differences in the importance of small ruminants in epidemiology of human cryptosporidiosis. These tools have also been used effectively in linking zoonotic transmission of C. parvum between outbreak cases and the suspected animals. Further studies should be directly elucidating the reasons for differences in the distribution and public health importance of major Cryptosporidium species in sheep and goats.

Cryptosporidiosis, caused by parasite species of the genus Cryptosporidium, is a major diarrhoeal disease in both people and animals globally, with C. hominis and C. parvum the main species infecting humans. Environmentally robust oocysts which are shed in high numbers in the faeces of infected individuals are resistant to disinfectants, including levels of chlorine normally used in drinking water. As a result, Cryptosporidium is a major cause of waterborne and foodborne outbreaks. Interestingly, C. hominis is responsible for the majority of waterborne outbreaks typed to date with C. parvum responsible for the majority of foodborne outbreaks. No vaccine and few treatments are currently available, which has greatly limited control of this disease to date. Livestock are both an important reservoir and source of human infections and improved husbandry and management practices as well as a One Health integrated molecular typing approach across both veterinary and public health systems are essential to improve our ability to control this disease.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 μm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

A new Eimeria species is described from a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia. Sporulated oocysts of Eimeria chalcoptereae n. sp. (n = 30) are subspheroidal, 22-25 × 21-24 (23.5 × 22.6) μm; length/width (L/W) ratio 1.0-1.1 (1.04) μm. Wall bi-layered, 1.0-1.4 (1.2) μm thick, outer layer smooth, c.2/3 of total thickness. Micropyle barely discernible. Oocyst residuum is absent, but 2 to 3 small polar granules are present. Sporocysts (n = 30) ellipsoidal, 13-14 × 7-8 (13.5 × 7.2) μm; L/W ratio 1.8-2.0 (1.88). Stieda body present, flattened to half-moon-shaped, 0.5 × 2.0 μm; sub-Stieda present, rounded to trapezoidal, 1.5 × 2.5 μm; para-Stieda body absent; sporocyst residuum present, usually as an irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with a robust refractile body and centrally located nucleus. Isolated Eimeria oocysts were analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. Analyses revealed that Eimeria chalcoptereae n. sp. shared the highest number of molecular features with an Eimeria sp. previously identified from a domestic pigeon in Australia (KT305927-29), with similarities at these three loci of 98.53%, 97.32% and 94.93%, respectively. According to morphological and molecular analysis, the isolated coccidian parasite is a new species of Eimeria named Eimeria chalcoptereae n. sp. after its host, the common bronzewing pigeon (Phaps chalcoptera) (Columbiformes: Columbidae) (Latham, 1790).

Cryptosporidium spp. are common protozoan pathogens in mammals. With pet rodents being integrated into modern life, the potential roles of them in transmitting parasites to humans need assessments. In the present study, we examined the occurrence of Cryptosporidium spp. in pet rodents in Guangdong, south China. A total of 697 fecal samples were collected from 11 species of rodents in seven pet shops, one pet market and one farm. Cryptosporidium spp. were identified by PCR analysis of the small subunit rRNA gene. An overall infection rate of 36.9% (257/697) was obtained, with infection rates varying from 9.3% in chinchillas, 52.3% in guinea pigs, 57.1% in squirrels, to 68.4% in cricetid animals. Nine Cryptosporidium species and genotypes were identified, including C. wrairi (in 129 guinea pigs), C. andersoni (in 34 hamsters), C. homai (in 32 guinea pigs), Cryptosporidium hamster genotype (in 30 hamsters), C. ubiquitum (in 24 chinchillas and squirrels), C. parvum (in 2 chinchillas), Cryptosporidium ferret genotype (in 2 chipmunks), C. muris (in 1 hamster and 1 guinea pig), and Cryptosporidium chipmunk genotype V (in 1 chinchilla and 1 chipmunk). Sequence analysis of the 60 kDa glycoprotein gene identified three subtype families of C. ubiquitum, including family XIId in 15 chinchillas, XIIa in 5 chinchillas, and a new subtype family (XIIi) in 1 squirrel. The identification of C. parvum and C. ubiquitum in pet rodents suggests that these animals, especially chinchillas, could serve as reservoirs of human-pathogenic Cryptosporidium spp. Hygiene should be practiced in the rear and care of these animals, and One Health measures should be developed to reduce the occurrence of zoonotic Cryptosporidium infections due to contact with pet rodents., Graphical abstract Unlabelled Image, Highlights • Cryptosporidium spp. were prevalent in pet rodents in Guangdong, China. • Nine Cryptosporidium species and genotypes were identified. • Chinchillas were commonly infected with zoonotic C. ubiquitum. • The XIId subtype family of C. ubiquitum has been imported into China together with chinchillas. • One Health measures should be developed to control zoonotic cryptosporidiosi.

Simple Summary Cryptosporidium is a parasite that infects humans and a broad range of animals. There are few diagnostic features that can be used to identify and differentiate between species and therefore DNA-based detection and genetic typing methods are required. This is important as some species are transmitted from animals to humans (a process called zoonotic transmission) and understanding this is central to control. These DNA-based tools have greatly facilitated our current understanding of which of the many Cryptosporidium species and genotypes that have been identified are capable of infecting humans. More studies need to be conducted in areas where the potential for zoonotic transmission is greatest and recently developed genetic tools should be applied more extensively to further our understanding of the zoonotic transmission of this important parasite. Abstract The enteric parasite, Cryptosporidium is a major cause of diarrhoeal illness in humans and animals worldwide. No effective therapeutics or vaccines are available and therefore control is dependent on understanding transmission dynamics. The development of molecular detection and typing tools has resulted in the identification of a large number of cryptic species and genotypes and facilitated our understanding of their potential for zoonotic transmission. Of the 44 recognised Cryptosporidium species and >120 genotypes, 19 species, and four genotypes have been reported in humans with C. hominis, C. parvum, C. meleagridis, C. canis and C. felis being the most prevalent. The development of typing tools that are still lacking some zoonotic species and genotypes and more extensive molecular epidemiological studies in countries where the potential for transmission is highest are required to further our understanding of this important zoonotic pathogen. Similarly, whole-genome sequencing (WGS) and amplicon next-generation sequencing (NGS) are important for more accurately tracking transmission and understanding the mechanisms behind host specificity.

The protozoan parasites Cryptosporidium and Giardia are significant causes of diarrhoea worldwide and are responsible for numerous waterborne and foodborne outbreaks of diseases. Over the last 50 years, the development of improved detection and typing tools has facilitated the expanding range of named species. Currently at least 44 Cryptosporidium spp. and >120 genotypes, and nine Giardia spp., are recognised. Many of these Cryptosporidium genotypes will likely be described as species in the future. The phylogenetic placement of Cryptosporidium at the genus level is still unclear and further research is required to better understand its evolutionary origins. Zoonotic transmission has long been known to play an important role in the epidemiology of cryptosporidiosis and giardiasis, and the development and application of next generation sequencing tools is providing evidence for this. Comparative whole genome sequencing is also providing key information on the genetic mechanisms for host specificity and human infectivity, and will enable One Health management of these zoonotic parasites in the future.

A new Caryospora-like isolate is described from a magpie-lark (Grallina cyanoleuca) in Western Australia. Sporulated oocysts of the Caryospora-like isolate (n = 35) are subspherical with a shape index of 1.13 ((21.5 (19.7-23.6) × 19.0 (18.1-19.8) μm). The bilayered oocyst wall is smooth. Micropyle, polar granule and oocyst residuum are absent. The sporocyst is ellipsoidal, 18.9 (17.2-20.8) × 12.3 (11.9-12.8) μm, with a shape index (length/width) of 1.54. The sporocyst wall is bilayered. Stieda and substieda bodies are present, the Stieda body is small and flattened and the substieda is trapezoidal. Sporocyst with eight sporozoites arranged head to tail. The sporozoites are vermiform, 18.9 (17.2-20.8) × 12.3 (11.9-12.8) μm and have striations at the anterior end. Each sporozoite has both anterior and posterior refractile bodies. A sporocyst residuum is present. Molecular characterization of the isolated Caryospora-like oocysts was conducted at the 18S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. At the 18S rRNA locus, the Caryospora-like isolate exhibited 88.8% to 96.5% similarity with other Caryospora spp. from different hosts. At the COI locus, it showed 91.5% similarity to Caryospora cf. bigenetica JB-2013 (KF859856) from the rattlesnake, Sistrurus catenatus.

Cryptosporidium species are a major cause of diarrhoea worldwide. In the present study, a retrospective analysis of 109 microscopically Cryptosporidium-positive faecal specimens from Western Australian patients, collected between 2015 and 2018 was conducted. Sequence analysis of the 18S rRNA and the 60 kDa glycoprotein (gp60) gene loci identified four Cryptosporidium species: C. hominis (86.2%, 94/109), C. parvum (11.0%, 12/109), C. meleagridis (1.8%, 2/109) and C. viatorum (0.9%, 1/109). Subtyping at the gp60 locus identified a total of 11 subtypes including the emergence of the previously rare C. hominis IfA12G1R5 subtype in 2017 as the dominant subtype (46.7%, 21/45). This subtype has also recently emerged as the dominant subtype in the United States but the reasons for its emergence are unknown. This is also the first report of C. viatorum in humans in Australia and a novel subtype (XVaA3g) was identified in the one positive patient.

Emerging infectious diseases are frequently zoonotic, often originating in wildlife, but enteric protozoa are considered relatively minor contributors. Opinions regarding whether pathogenic enteric protozoa may be transmitted between wildlife and humans have been shaped by our investigation tools, and have led to oscillations regarding whether particular species are zoonotic or have host-adapted life cycles. When the only approach for identifying enteric protozoa was morphology, it was assumed that many enteric protozoa colonized multiple hosts and were probably zoonotic. When molecular tools revealed genetic differences in morphologically identical species colonizing humans and other animals, host specificity seemed more likely. Parasites from animals found to be genetically identical - at the few genes investigated - to morphologically indistinguishable parasites from human hosts, were described as having zoonotic potential. More discriminatory molecular tools have now sub-divided some protozoa again. Meanwhile, some infection events indicate that, circumstances permitting, some “host-specific” protozoa, can actually infect various hosts. These repeated changes in our understanding are linked intrinsically to the investigative tools available. Here we review how molecular tools have assisted, or sometimes confused, our understanding of the public health threat from nine enteric protozoa and example wildlife hosts (Balantoides coli - wild boar; Blastocystis sp. - wild rodents; Cryptosporidium spp. - wild fish; Encephalitozoon spp. - wild birds; Entamoeba spp. - non-human primates; Enterocytozoon bieneusi - wild cervids; Giardia duodenalis - red foxes; Sarcocystis nesbitti - snakes; Toxoplasma gondii - bobcats). Molecular tools have provided evidence that some enteric protozoa in wildlife may infect humans, but due to limited discriminatory power, often only the zoonotic potential of the parasite is indicated. Molecular analyses, which should be as discriminatory as possible, are one, but not the only, component of the toolbox for investigating potential public health impacts from pathogenic enteric protozoa in wildlife., Graphical abstract Image 1, Highlights • Wildlife is a potential reservoir of disease agents that may infect humans. • The public health threat from enteric protozoa in wildlife is poorly understood. • Molecular tools may help in understanding this threat, but may also confuse. • We use nine enteric protozoa-wildlife host examples to review the current position. • Data accumulate, but more discriminatory tools and other approaches are important.

A new Isospora (Apicomplexa: Eimeriidae) species is described from an Australian raven (Corvus coronoides) in Western Australia. Sporulated oocysts (n = 21) are ovoid, 21.2 (18.4-23.9) μm in length and 18.8 (16.9-20.6) μm in width, with a shape index of 1.13. The bi-layered oocyst wall is smooth and colourless, 1.2 μm thick. A polar granule and oocyst residuum is present, but the micropyle is absent. The sporocysts are ovoid-shaped, 16.3 (13.7-18.9) × 10.7 (8.4-12.9) μm, with a shape index (length/width) of 1.52. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda being indistinct. Each sporocyst with four vermiform sporozoites arranged head to tail. The sporozoites are crescent-shaped, 9.0 (8.9-9.2) × 2.7 (2.3-3.0) μm, with a shape index (length/width) of 3.33. The sporocyst residuum is present. The isolated oocysts had different morphological characteristics when compared with all known Isospora spp. The coccidian parasite was analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. At the 18S locus, I. coronoideae n. sp. exhibited 98.9% similarity to I. neochmiae from a captive-bred red-browed finch (KT224380) and Isospora sp. from domestic pigeons (Columba livia domestica) (AB757860), 98.5% similarity to I. gryphoni (AF080613) from an American goldfinch and 98.3% similarity to I. manorinae (KT224379) from a yellow-throated miner. At the 28S locus, it exhibited 95.4% and 94.8% similarity to I. manorinae (KT224381) and I. anthochaerae (KF766053), respectively. At the COI locus, it exhibited 99.8% and 99.7% similarity to I. butcherae (KY801687) and I. neochmiae (KT224378), respectively. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora coronoideae n. sp. after its host, the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758).

Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gammaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species.

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. 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). 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. 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.

A new Isospora species is described from New Holland honeyeaters (Phylidonyris novaehollandiae). Sporulated oocysts (n = 25) were characterised as subspheroidal, 29–32 × 28–31 (29.8 × 29.4); length/width (L/W) ratio 1.01–1.02 (1.01). Wall bi-layered, 1.3–1.6 (1.5) thick, outer layer smooth, c.2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 25) ovoidal, 18–19 × 12–14 (18.4 × 12.3); L/W ratio 1.42–1.53 (1.50). Stieda body present, flattened, c.0.5 deep × 2.5 wide; sub-Stieda present, rounded, c.2.5 deep × 3.5 wide; para-Stieda body absent; sporocyst residuum present, usually a distinctly irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with robust anterior and posterior refractile bodies. Molecular characterization was conducted at the 18S and 28S ribosomal RNA and the mitochondrial (mt) cytochrome oxidase (COI) loci. Phylogenetic analysis of genomic 18S and mt COI sequences indicated that Isospora phylidonyrisae n. sp. was genetically similar to Isospora coronoideae, isolated from an Australian raven (Corvus coronoides) in Western Australia, with a 99.3% and 98.4% homology, respectively. The 28S rRNA sequence was most similar to Isospora anthochaerae (KF766053) and Isospora manorinae (KT224381), both with a 98.2% genetic similarity. Based on morphological and genetic data, this isolate is a new species of Isospora, which is named Isospora phylidonyrisae n. sp. after its host.

The use of molecular tools has led to the identification of several zoonotic Cryptosporidium spp. in dogs and cats. Among them, Cryptosporidium canis and Cryptosporidium felis are dominant species causing canine and feline cryptosporidiosis, respectively. Some Cryptosporidium parvum infections have also been identified in both groups of animals. The identification of C. canis, C. felis and C. parvum in both pets and owners suggests the possible occurrence of zoonotic transmission of Cryptosporidium spp. between humans and pets. However, few cases of such concurrent infections have been reported. Thus, the cross-species transmission of Cryptosporidium spp. between dogs or cats and humans has long been a controversial issue. Recently developed subtyping tools for C. canis and C. felis should be very useful in identification of zoonotic transmission of both Cryptosporidium spp. Data generated using these tools have confirmed the occurrence of zoonotic transmission of these two Cryptosporidium spp. between owners and their pets, but have also shown the potential presence of host-adapted subtypes. Extensive usage of these subtyping tools in epidemiological studies of human cryptosporidiosis is needed for improved understanding of the importance of zoonotic transmission of Cryptosporidium spp. from pets.

A new coccidian species, Isospora lugensae n. sp., was described from a single Kerguelen petrel (Lugensa brevirostris). Sporulated oocysts (n = 25) were characterized as subspheroidal to ellipsoidal measuring 24–25 μm × 21–23 μm (24.8 × 22.2 μm) in length/width (L/W), respectively, with a ratio of 1.07–1.14 μm (1.12). They contained a bi-layered wall with a thickness of 0.8–1.2 μm (1.0) and the outer layer smooth, with c.2/3 of total thickness. The oocyst contained two polar granules with both micropyle and oocyst residuum absent. Ovoidal sporocysts (n = 25) measured 15–16 μm × 10–11 μm (15.7 × 10.8 μm) in L/W, with a ratio of 1.41–1.49 μm (1.46). A flattened to knob-like Stieda body was present (c.0.5 μm deep × 2.5 μm wide) as well as a rounded to trapezoidal sub-Stieda (c.1.5 μm deep × 3.0 μm wide); however, no para-Stieda body was detected. The sporocyst residuum was composed of scattered spherules of different sizes, while vermiform sporozoites contained a refractile body, nucleus and visible striations. Analysis of the full-length mitochrondrial (mtDNA) genome revealed 3 protein-coding genes, (CytB, COI and COIII), 18 LSU and 14 small subunit (SSU) rDNA fragments, without transfer RNA genes with a total length of 6257 bp. Phylogenetic analysis of genomic SSU ribosomal sequences indicated that Isospora lugensae n. sp. is genetically similar to Eimeria reichenowi, isolated from a red-crowned crane (Grus japonensis) from Japan, with a 96.6% homology. The mtDNA sequence is most similar to Isospora serinuse with a 95.8% genetic similarity. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in a Kerguelen petrel.

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.

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.

Trypanosomes are blood-borne parasites that can infect a variety of different vertebrates, including animals and humans. This study aims to broaden scientific knowledge about the presence and biodiversity of trypanosomes in Australian bats. Molecular and morphological analysis was performed on 86 blood samples collected from seven different species of microbats in Western Australia. Phylogenetic analysis on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences identified Trypanosoma dionisii in five different Australian native species of microbats; Chalinolobus gouldii, Chalinolobus morio, Nyctophilus geoffroyi, Nyctophilus major and Scotorepens balstoni. In addition, two novels, genetically distinct T. dionisii genotypes were detected and named T. dionisii genotype Aus 1 and T. dionisii genotype Aus 2. Genotype Aus 2 was the most prevalent and infected 20.9% (18/86) of bats in the present study, while genotype Aus 1 was less prevalent and was identified in 5.8% (5/86) of Australian bats. Morphological analysis was conducted on trypomastigotes identified in blood films, with morphological parameters consistent with trypanosome species in the subgenus Schizotrypanum. This is the first report of T. dionisii in Australia and in Australian native bats, which further contributes to the global distribution of this cosmopolitan bat trypanosome.

Current knowledge of Cryptosporidium species/genotypes in marine fish is limited. Following phylogenetic analysis at the 18S rDNA locus, a recent study identified six new genotypes of Cryptosporidium colonizing edible fish found in European seas. Of these, five grouped in a clade together (#Cryptofish 1&ndash, 5) and one grouped separately (#Cryptofish 7). In the present study, after phylogenetic analyses of #Cryptofish1, #Cryptofish2, #Cryptofish4, #Cryptofish5 and #Cryptofish7 at the actin locus, the presence of two major clades was confirmed. In addition, when possible, longer 18S amplicons were generated. In conclusion, the small genetic distances between these genotypes designated as a novel marine genotype I (#Cryptofish 1-5) suggest that they may be genetic variants of the same species, while the designated novel marine genotype 2 (#Cryptofish 7) is clearly representative of a separate species.

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert's potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.

Natural landscape alterations as a consequence of urbanisation are one of the main drivers in the movements of wildlife into metropolitan and peri-urban areas. Worldwide, these wildlife species are highly adaptable and may be responsible for the transmission of tick-borne pathogens including piroplasms (Babesia, Theileria and Cytauxzoon spp.) that cause piroplasmosis in animals and occasionally in humans. Little is known about piroplasms in the ticks of urban wildlife in Australia. Ticks from long-nosed bandicoots (Perameles nasuta; n = 71), eastern-barred bandicoots (Perameles gunnii; n = 41), northern-brown bandicoots (Isoodon macrourus; n = 19), southern-brown bandicoots (Isoodon obesulus; n = 4), bandicoot sp. (n = 2), flying foxes (Pteropus sp.; n = 3), black rats (Rattus rattus; n = 7), bush rats (Rattus fuscipes; n = 4), brushtail possums (Trichosurus vulpecula; n = 19), ringtail possums (Pseudocheirus peregrinus; n = 12), short-eared possums (Trichosurus caninus; n = 6), possum sp. (Trichosurus sp.; n = 8), and red foxes (Vulpes vulpes; n = 12) were analysed using piroplasm-specific 18S primers and Sanger sequencing. Seven Ixodes tasmani ticks from long-nosed bandicoots and bandicoots sp., three I. tasmani ticks and one Ixodes holocyclus tick from brushtail possums, and one Haemaphysalis longicornis tick from a red fox were positive for piroplasms. New genotypes, with sequences sharing 98% nucleotide similarities with Theileria sp. K1 detected in a burrowing bettong (Bettongia lesueur), were identified from bandicoot ticks. New genotypes were detected in ticks from brushtail possums, which shared 98% similarity with a Babesia sp. (JQ682877) previously identified in marsupials. Theileria orientalis was identified in the H. longicornis tick from the red fox. Babesia and Theileria spp. in the ticks parasitizing bandicoots and brushtail possums clustered closely with respective Babesia and Theileria clades derived from Australian marsupials. This represents the first detection of piroplasms in ticks parasitizing brushtail possums and a red fox in Australia., Graphical abstract Image 1, Highlights • First characterisation of novel Babesia sp. in ticks from brushtail possums in Australia. • First detection of Theileria orientalis in tick on a red fox in Australia. • Theileria sp. derived from Australian marsupials form a unique clade.

To identify the gastrointestinal helminths of veterinary, zoonotic and public health importance in farmers and their ruminant livestock in Ghana, faecal samples were collected from 95 farmers and their livestock (cattle = 328, sheep = 285 and goats = 217) and examined by microscopy and/or molecular techniques. Overall, 21 farmers tested positive for at least one gastrointestinal helminth, 80.9% of which were single infections and 19.0% co-infections. The parasites identified in the farmers consisted of hookworms (n = 13) (9 were Necator americanus and the other 4 could not be amplified by PCR), Trichostrongylus spp. (n = 9), Schistosoma mansoni (n = 1), Schistosoma haematobium (n = 1) and Diphyllobothrium latum (n = 1). In livestock, strongylid nematodes were dominant (56.6%), followed by Paramphistomum spp. (16.9%), Dicrocoelium spp. (7.1%), Thysaniezia spp. (5.8%), Trichuris spp. (3.3%), Moniezia spp. (3.1%), Fasciola spp. (2.8%), Toxocara spp. (1.1%) and Schistosoma spp. (0.2%). Genotyping of Trichostrongylus spp. in the farmer's stools identified six T. colubriformis similar to T. colubriformis detected in cattle, sheep and goats in the study, two Trichostrongylus spp. with 98.3% and 99.2% genetic similarity to T. probolurus respectively and one Trichostrongylus spp. which showed 96.6% similarity to both T. probolurus and T. rugatus. Trichostrongylus axei was also identified in cattle, sheep and goats. This is the first molecular characterisation of Trichostrongylus spp. in Ghana and the species identified in the present study suggests zoonotic transmission from cattle, sheep and goats. Further studies involving larger numbers of farmers and their household members are essential to understand the transmission dynamics and impact of these parasites on farming communities in Ghana.

A survey was conducted on 30 Danish mink farms from April to October 2016 to determine the prevalence and species of Eimeria in Danish farmed mink. In total, 2.6% of mink faecal samples (108/4140) were positive for Eimeria vison-like oocysts by microscopy, with 24.8% (78/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts (n = 20) identified Eimeria vison-like oocysts measuring 21.0 × 13.8 μm with a length/width (L/W) ratio of 1.5. Phylogenetic analysis of 18S rRNA sequences (1221 bp) from three positive mink indicated that Eimeria vison-like shared the highest genetic similarity to Eimeria sp. ex Apodemus agrarius from a Striped field mouse (A. agrarius) from the Czech Republic (99.6%). Analysis of a shorter region of 18S (531 bp) revealed that the E. vison-like genotype sequences grouped in the same clade and shared 97.7% similarity with E. furonis. At the cytochrome c oxidase subunit I (COI) locus, mink-derived sequences were not available from GenBank and phylogenetic analysis placed the novel E. vison-like in a clade with E. cf. ictidea (99.4% similarity) from a black footed ferret (Mustela nigripes) from Canada.

Associations between faecal shedding of pathogenic Yersinia enterocolitica (based on the yst virulence gene) with growth, carcass weight and diarrhoea were investigated using an observational longitudinal study of 1200 crossbred prime (meat) lambs on eight Australian farms. Live weight, breech faecal soiling score (scale 1-5) and faecal consistency score (FCS; scale 1-5) were recorded, and faecal samples collected from each lamb on three sampling occasions; weaning (≈12 weeks of age), post-weaning (≈19 weeks) and pre-slaughter (≈29 weeks). Hot standard carcass weight was measured at slaughter. Faecal samples were screened for presence and concentration of pathogenic Y. enterocolitica using quantitative PCR. Associations of pathogenic Y. enterocolitica detection and shedding intensity with lamb health and production were assessed using general linear models (carcass weight), linear mixed effects models (live weight, FCS and breech soiling score) and non-parametric tests (FCS and breech soiling score). Prevalence for non-pelleted faeces (FCS ≥ 3.0) and diarrhoea (FCS ≥ 4.0) were compared with the two-tailed z-test, odds ratios and relative risk. Lambs shedding pathogenic Y. enterocolitica were 3.78 kg lighter post-weaning (P < 0.001) and 2.61 kg lighter pre-slaughter (P = 0.035) compared to lambs in which pathogenic Y. enterocolitica was not detected. Higher faecal concentration of pathogenic Y. enterocolitica was associated with lower live weight (P < 0.001). There was no association between pathogenic Y. enterocolitica detection and carcass weight. Overall, there was no evidence of association between pathogenic Y. enterocolitica detection and diarrhoea (higher FCS, higher risk for non-pelleted faeces or diarrhoea, or higher breech soiling score). Only one flock had increased relative risk for non-pelleted faeces associated with pathogenic Y. enterocolitica detection, and one other flock had increased relative risk for diarrhoea associated with pathogenic Y. enterocolitica detection. This is the first report of an association between reduced sheep live weight and pathogenic Y. enterocolitica based on the presence of the yst gene for heat stable enterotoxin determined by qPCR in sheep. Notably, impacts on live weight were observed in the absence of diarrhoea.

A new Isospora (Apicomplexa:Eimeriidae) species is described from a silvereye (Zosterops lateralis) in Western Australia. Sporulated oocysts of this species are spherical, 24.2 (23.1-25.2) × 23.3 (22.8-23.9) μm, with a shape index (length/width) of 1.02, and with a smooth bi-layered oocyst wall, 1.2 μm thick (outer layer 0.9 μm, inner 0.3 μm). A polar granule is present, but the oocyst residuum and a micropyle are absent. The ovoid-shaped sporocysts are 16.1 (15.7-17.3) × 10.5 (15.7-17.3) μm and have a shape index of 1.53. A hemidome-shaped Stieda and a rectangular-shaped substieda body are present. A sporocyst residuum is present and composed of numerous granules of different sizes scattered among the sporozoites. The oocysts from this isolate are morphologically different from those of all known Isospora spp. This coccidian parasite was molecularly characterised at the 18S, 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, based on 1210 bp of sequence, this new isolate exhibited 99.9, 99.8, 99.7 and 99.5% similarity to I. sp. MAH-2013a (KF648870) from a superb starling (Lamprotornis superbus) in Canada, I. sp. MS-2003 (AY33157) from a Southern cape sparrow (Plocepasser mahali) in America, I. sp. Tokyo (AB75786) from Japan and I. sp. respectively. Further analysis of a subgroup of 300 bp long 18S sequences (n = 11), including I. anthochaerae and the other three Isospora characterised from birds in Western Australia, revealed that I. butcherae n. sp. exhibited 98.3% similarity to both I. sp. MAH-2013a (KF648870) and I. MS-2003 (AY33171). At the 28S locus, this new isolate exhibited 97.3% similarity with I. sp. MS-2003 from a California towhee (Melozone crissalis). At the COI locus, this new isolate exhibited 99.8% similarity to I. neochmiae from a red-browed finch. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora butcherae n. sp. after Mrs. June Butcher for her lifelong dedication as a wildlife rehabilitator. Graphical abstract ᅟ.

Worldwide, Ehrlichia spp. are emerging infectious organisms of domestic animals and people, however, most Ehrlichia spp. naturally infect wildlife reservoirs causing mainly asymptomatic infections. Australian ecosystems have been under-explored for these potentially pathogenic organisms, and recent studies have identified a range of novel Ehrlichia, and their sister genera, Anaplasma and 'Candidatus Neoehrlichia' species, from native Australian ticks. We used bacterial 16S rRNA (16S) next-generation sequencing and genus-specific PCR to profile the bacterial communities in platypus (Ornithorhynchus anatinus) blood samples and platypus ticks (Ixodes ornithorhynchi), and identified a high prevalence of Ehrlichia sequences. We also observed Ehrlichia-like intra-neutrophilic inclusions (morulae) in PCR-positive stained platypus blood films that were consistent in morphology with other Ehrlichia spp. Bayesian phylogenetic analysis of 16S (1343 bp), gltA (1004 bp), and groEL (1074 bp) gene sequences group the platypus Ehrlichia with 'Candidatus Ehrlichia khabarensis' from far-eastern Russia, and demonstrate that the platypus Ehrlichia is clearly distinct from all other Ehrlichia spp. Enough genetic divergence exists to delineate this platypus Ehrlichia as a separate species that we propose to designate 'Candidatus Ehrlichia ornithorhynchi'. There is no evidence that 'Candidatus Ehrlichia ornithorhynchi' causes disease in wild platypuses, however, the organism does seem to be widespread in Australia, being found in both Queensland and Tasmania. 'Candidatus Ehrlichia ornithorhynchi' is the second native Australian Ehrlichia described and adds to the rapidly growing diversity of recently described native Australian tick-borne bacteria.

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data.

Western Australian rangeland goats were surveyed for faecal carriage of Salmonella enterica and Campylobacter spp. Faecal samples were collected from 125 goats on four occasions. The first sample was collected immediately upon arrival at a commercial goat depot (feedlot). Subsequent samples were collected at one month intervals thereafter. Frequency of detection and faecal carriage intensity were determined using qPCR targeting the S. enterica outer membrane protein (ompF) and Campylobacter spp. purine biosynthesis gene (purA). Salmonella enterica were identified in 40/500 of faecal samples, with S. enterica faecal carriage detected in 30% (38/125) goats over the duration of the study. Campylobacter spp. were identified in 12/500 of samples, with Campylobacter spp. detected in 10% (12/125) goats over duration of the study. Frequency of detection was highest at the first sample collection for both S. enterica (26%) and Campylobacter spp. (8%). Repeat detection of Salmonella was observed for only a single goat (0.8%). Salmonella qPCR positive samples were characterised at ompF and invA genes as S. enterica. Further characterisation at STM2755 and STM4497 genes confirmed the isolates were S. enterica serovar Typhimurium. Characterization at the 16S rRNA and hippuricase (hipO) genes revealed all Campylobacter spp. positive samples were C. jejuni. This study demonstrates that qPCR can be used for rapid identification of faecal carriage in goat faecal samples and showed evidence of carriage of zoonotic S. Typhimurium and C. jejuni by captured rangeland goats. The findings have implications for management of goats at abattoirs and in confined feeding facilities.

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 and Giardia are major causes of diarrhoea in developing countries including Ghana, however, nothing is known about the species and subtypes of Cryptosporidium and Giardia in farmers and their ruminant livestock in this country. A total of 925 faecal samples from humans (n = 95), cattle (n = 328), sheep (n = 217) and goats (n = 285), were screened for Cryptosporidium and Giardia by quantitative PCR (qPCR) at the 18S rRNA and glutamate dehydrogenase (gdh) loci respectively. Cryptosporidium positives were typed by sequence analysis of 18S and 60 kDa glycoprotein (gp60) loci amplicons. Giardia positives were typed at the triose phosphate isomerase (tpi), beta-giardin (bg) and gdh loci. The prevalence of Cryptosporidium and Giardia by qPCR was 8.4% and 10.5% in humans, 26.5% and 8.5% in cattle, 34.1% and 12.9% in sheep, and 33.3% and 12.3% in goat faecal samples, respectively. G. duodenalis assemblages A and B were detected in humans and assemblage E was detected in livestock. Cryptosporidium parvum was the only species identified in humans; C. andersoni, C. bovis, C. ryanae and C. ubiquitum were identified in cattle; C. xiaoi, C. ubiquitum and C. bovis in sheep; and C. xiaoi, C. baileyi and C. parvum in goats. This is the first molecular study of Cryptosporidium and Giardia in livestock in Ghana. The identification of zoonotic species and the identification of C. parvum subtype IIcA5G3q in livestock, which has previously been identified in children in Ghana, suggests potential zoonotic transmission. Further studies on larger numbers of human and animal samples, and on younger livestock are required to better understand the epidemiology and transmission of Cryptosporidium and Giardia in Ghana.

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, disease severity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing. The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n=11), Cryptosporidium parvum (n=22) and Cryptosporidium cuniculus (n=8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n=11), four C. parvum subtypes belonging to IIa (n=3) and IId (n=19) and one C. cuniculus subtype (VbA23) (n=8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies.

Faecal shedding of Eimeria by captured rangeland goats (Capra hircus) was investigated using a longitudinal observational study. Faecal samples were collected from 125 male goats on four occasions. The first sampling occurred following capture and transport, immediately after arrival at a commercial goat depot (feedlot) in Western Australia, with subsequent 3 sample collections occurring at one month intervals thereafter. Goats were composite breed and aged approximately 9–12 months on arrival at the feedlot. Prevalence and shedding intensity (faecal oocyst concentration) for Eimeria were determined using qPCR. Species were identified from individual oocysts (isolated using micromanipulation) using molecular analysis at two loci, specifically 18S rRNA and mitochondrial cytochrome oxidase gene (COI), and confirmed by microscopy. Longitudinal prevalence (animals positive at least once) for Eimeria spp. by qPCR was 90.4%, with 60% goats shedding Eimeria spp. on more than one occasion. Point prevalence (prevalence at a single sampling occasion) ranged from 2.4% (fourth sampling) to 70.4% (second sampling). Three species were identified at the 18S rRNA locus and confirmed by microscopy: E. christenseni (longitudinal prevalence for single infection 34.4%), E. hirci (17.6%) and E. arloingi (8.8%) over the four sample collections. Mixed infections were identified in 56.8% goats (longitudinal prevalence). 18S rRNA sequences from E. christenseni and E. hirci were 100% homologous with ovine E. ahsata and E. crandallis respectively, and E. arloingi was 100% similar to caprine E. arloingi. At the COI locus, E. christenseni, E. hirci and E. arloingi grouped separately, and were closely related to ovine E. ahsata, with genetic similarities of 96.5%, 92.6% and 91.4% respectively. This is the first report for molecular characteristics of caprine-derived Eimeria spp. using a combination of 18S rRNA and COI. Molecular techniques can be used to identify Eimeria spp. in goat faecal samples, specifically through characterization at 18S locus and other gene loci when used in parallel. Molecular techniques offer some advantages over microscopy for identification of Eimeria species, particularly with respect to precision.

Anaplasma and Ehrlichia spp. are tick-borne pathogens that can cause severe disease in domestic animals, and several species are responsible for emerging zoonoses in the northern hemisphere. Until recently, the only members of these genera reported in Australia (A. marginale, A. centrale, and A. platys) were introduced from other continents, through the importation of domestic animals and their associated ticks. However, unique Anaplasma and Ehrlichia 16S rRNA gene sequences were recently detected for the first time in native Australian ticks, particularly in Amblyomma triguttatum subsp. ticks from southwest Western Australia (WA). We used molecular techniques to survey Am. triguttatum subsp. ticks from four allopatric populations in southern and western Australia for Anaplasma and Ehrlichia species, and described here the phylogeny of these novel organisms. An A. bovis variant (genotype Y11) was detected in ticks from two study sites; Yanchep National Park (12/280, 4.3%) and Barrow Island (1/69, 1.4%). Phylogenetic analysis of 16S rRNA and groEL gene sequences concluded that A. bovis genotype Y11 is a unique genetic variant, distinct from other A. bovis isolates worldwide. Additionally, a novel Ehrlichia species was detected in Am. triguttatum subsp. from three of the four study sites; Yanchep National Park (18/280, 6.4%), Bungendore Park (8/46, 17.4%), and Innes National Park (9/214, 4.2%), but not from Barrow Island. Phylogenetic analysis of 16S, groEL, gltA, and map1 gene sequences revealed that this Ehrlichia sp. is most closely related to, but clearly distinct from, E. ruminantium and Ehrlichia sp. Panola Mountain. We propose to designate this new species '. Candidatus Ehrlichia occidentalis'. Anaplasma bovis genotype Y11 and 'Candidatus E. occidentalis' are the first Anaplasma and Ehrlichia species to be recorded in native Australian ticks. Refereed/Peer-reviewed

Enteric parasites are major contributors to the global diarrhoeal disease load, infecting >67.2 million people. Their prevalence and clinical impact, however, are underestimated due to lack of adequate detection, which is largely still based on microscopy, particularly in developing countries. New commercially available enteric panel assays, which detect parasites (as well as bacteria and/or viruses) using multiplex PCR, offer enhanced sensitivity and specificity as well as the ability to detect mixed infections, and will play an important role in epidemiological surveillance and outbreak investigations. A major limitation of these technologies, however, particularly for developing countries, is the costs involved. Emerging technologies for low-resource, point-of-care (POC) settings have the potential to dramatically improve the cost and accuracy of enteric parasite detection in the future.

Chlamydiae are obligate intracellular bacteria that infect a broad range of host species, including sheep. Two species of Chlamydia infect sheep, C. abortus, which is a major cause of abortion in both sheep and goats, and C. pecorum, which causes pneumonia, arthritis/polyarthritis, encephalomyelitis, conjunctivitis, enteritis, abortion and metritis and infertility in domestic ruminants and pigs. The prevalence of faecal shedding of C. pecorum is relatively common amongst lambs in Australia. The aim of the work presented here was to use qPCR to determine the prevalence of C. pecorum in synovial samples obtained from abnormal joints trimmed from lamb carcases at one abattoir in southern Australia. The study included 53,131 carcases screened for arthritis, of which 369 had at least one abnormal joint trimmed. One hundred and forty eight trimmed joints were undamaged and suitable for PCR testing. The prevalence of C. pecorum in synovial tissue collected from the abnormal joints was 6.1% and the bacterial concentration ranged from 6 × 103 to 7.6 × 105/g of synovial tissue. Five of the positive joint samples were from carcases that had one joint trimmed for arthritis and four were from carcases from which two joints had been trimmed. None of the carcases from which the positive joint samples originated were condemned. The average arthritis trim weight of the carcases from which synovial tissue tested positive for C. pecorum was 1.112 kg (95% confidence interval 0.637-1.586 kg) and this did not differ from the carcases from which synovial tissue was not positive for C. pecorum. (mean 0.997 kg, 95% confidence interval 0.840-1.154 kg). Further research is required to determine the on-farm production losses associated with C. pecorum infection in Australian lambs.

Objective Develop a multiplex quantitative PCR assay to investigate the prevalence and shedding of Escherichia coli O157/O145, Salmonella spp. and Campylobacter spp. in sheep at sale yards and abattoirs. Methods A qPCR for E. coli O157/O145 was developed, validated and multiplexed with an existing qPCR for Campylobacter and Salmonella enterica. The absolute numbers of E. coli O157/O145, Campylobacter and Salmonella in control samples was determined using droplet digital PCR. These were then used as the controls in the multiplex qPCR on a total of 474 sheep faecal samples collected from two saleyards over a 4-month period (April–July 2014) and 96 effluent samples from an abattoir. Results The mutiplex qPCR was specific with a sensitivity of 5 organisms/μL faecal DNA extract for Campylobacter, S. enterica and E. coli O157/O145. The overall prevalence of Campylobacter, S. enterica and E. coli O157/O145 in faecal samples was 5.7%, 3.6% and 8.4% and in effluent samples was 18.8%, 6.3% and 5.2%, respectively. The pathogen loads of Campylobacter, S. enterica and E. coli O157/O145 in faecal and effluent samples was also determined via mutiplex qPCR. Conclusions The overall prevalences of Campylobacter, S. enterica and E. coli O157/O145 were generally low (

Cryptosporidium and Giardia are important causes of diarrhoeal illness. Adequate knowledge of the molecular diversity and geographical distribution of these parasites and the environmental and climatic variables that influence their prevalence is important for effective control of infection in at-risk populations, yet relatively little is known about the epidemiology of these parasites in Africa. Cryptosporidium is associated with moderate to severe diarrhoea and increased mortality in African countries and both parasites negatively affect child growth and development. Malnutrition and HIV status are also important contributors to the prevalence of Cryptosporidium and Giardia in African countries. Molecular typing of both parasites in humans, domestic animals and wildlife to date indicates a complex picture of both anthroponotic, zoonotic and spill-back transmission cycles that requires further investigation. For Cryptosporidium, the only available drug (nitazoxanide) is ineffective in HIV and malnourished individuals and therefore more effective drugs are a high priority. Several classes of drugs with good efficacy exist for Giardia, but dosing regimens are suboptimal and emerging resistance threatens clinical utility. Climate change and population growth are also predicted to increase both malnutrition and the prevalence of these parasites in water sources. Dedicated and co-ordinated commitments from African governments involving “One Health” initiatives with multidisciplinary teams of veterinarians, medical workers, relevant government authorities, and public health specialists working together are essential to control and prevent the burden of disease caused by these parasites.

Uninucleated Entamoeba cysts measuring 7.3 × 7.7 μm were detected in faecal samples collected from wild Rangeland goats (Capra hircus) after arrival at a commercial goat depot near Geraldton, Western Australia at a prevalence of 6.4% (8/125). Sequences were obtained at the 18S rRNA (n = 8) and actin (n = 5) loci following PCR amplification. At the 18S locus, phylogenetic analysis grouped the isolates closest with an E. bovis isolate (FN666250) from a sheep from Sweden with 99% similarity. At the actin locus, no E. bovis sequences were available, and the isolates shared 94.0% genetic similarity with E. suis from a pig in Western Japan. This is the first report to describe the morphology and molecular characterisation of Entamoeba from Rangeland goats in Western Australia and the first study to produce actin sequences from E. bovis-like Entamoeba sp.

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

Invasive rodent species are known hosts for a diverse range of infectious microorganisms and have long been associated with the spread of disease globally. The present study describes molecular evidence for the presence of a Trypanosoma sp. from black rats (Rattus rattus) in northern Sydney, Australia. Sequences of the 18S ribosomal RNA (rRNA) locus were obtained in two out of eleven (18%) blood samples with subsequent phylogenetic analysis confirming the identity within the Trypanosoma lewisi clade.

Background Apicomplexan parasites of the genus Cryptosporidium infect a wide range of animal species as well as humans. Cryptosporidium spp. can cause life threatening diarrhea especially in young animals, children, immunocompromised patients and malnourished individuals. Asymptomatic cryptosporidial infections in animals can also occur, making these animals potential reservoirs of infection. Methods In the present study, a molecular survey of Cryptosporidium spp. in ruminants that were slaughtered for human consumption in Yazd Province, located in central Iran was conducted. Faeces were collected per-rectum from 484 animals including 192 cattle, 192 sheep and 100 goats. DNA was extracted from all samples and screened for Cryptosporidium by PCR amplification of the 18S rRNA gene. Positives were Sanger sequenced and further subtyped by sequence analysis of the 60 kDa glycoprotein (gp60) locus. Results In total, Cryptosporidium spp. were detected in 22 animals: C. andersoni and C. bovis in seven and two cattle faecal samples, respectively, C. ubiquitum in five sheep, and C. xiaoi in six sheep and two goat samples, respectively. To our knowledge, this study provides for the first time, molecular information concerning Cryptosporidium species infecting goats in Iran, and is also the first report of C. ubiquitum and C. xiaoi from ruminants in Iran. Conclusion The presence of potentially zoonotic species of Cryptosporidium in ruminants in this region may suggest that livestock could potentially contribute to human cryptosporidiosis, in particular among farmers and slaughterhouse workers, in the area. Further molecular studies on local human populations are required to more accurately understand the epidemiology and transmission dynamics of Cryptosporidium spp. in this region.

The Bellinger River snapping turtle (Myuchelys georgesi) is endemic to Australia and is confined to a highly restricted distribution in the Bellinger River in New South Wales. Routine veterinary health examinations of 17 healthy turtles were undertaken, along with the collection and analysis of blood samples, during conservation efforts to save the species following a catastrophic population decline. Microscopy analysis of blood films detected Haemoproteidae parasites that morphologically resembled Haemocystidium chelodinae inside turtle erythrocytes. Of the 17 turtles examined, 16 were positive for infection with H. chelodinae by both light microscopy and PCR. DNA sequencing of a partial fragment of the mitochondrial cytochrome b (cytb) gene and phylogenetic analysis identified two different H. chelodinae-like genotypes. The phylogenetic relationship of H. chelodinae-like to other Haemoproteidae species based on cytb sequences grouped H. chelodinae-like into the reptile clade, but revealed the Haemocystidium genus to be paraphyletic as the clade also contained Haemoproteus, thus supporting a re-naming of Haemoproteus species from reptiles to Haemocystidium species. This study reports for the first time the genetic characterisation of H. chelodinae-like organisms isolated from a new Testudine host species, the Bellinger River snapping turtle. As evidence grows, further research will be necessary to understand the mode of transmission and to investigate whether these parasites are pathogenic to their hosts.

International audience; Cryptosporidium, a zoonotic pathogen, is able to infect a wide range of hosts including wild and domestic animals, and humans. Although it is well known that some parasites are both fish pathogens and recognized agents of zoonosis with a public health impact, little information is available concerning the prevalence of Cryptosporidium in wild aquatic environments. To evaluate the prevalence of Cryptosporidium spp. in commercially important edible marine fish in different European seas (English channel, North sea, Bay of Biscay, Celtic sea and Mediterranean sea), 1,853 specimens were collected as part of two surveys. Nested PCR followed by sequence analysis at the 18S rRNA gene locus was used to identify Cryptosporidium spp. The overall prevalence of Cryptosporidium spp. in sampled fish reached 2.3% (35 out of 1,508) in a first campaign and 3.2% (11 out of 345) in a second campaign. Sequence and phylogenetic analysis of positive samples identified Cryptosporidium parvum (n = 10) and seven genotypes which exhibited between 7.3 and 10.1% genetic distance from C. molnari, with the exception of one genotype which exhibited only 0.5-0.7% genetic distance from C. molnari. Among 31 analyzed fish species, 11 (35.5%) were identified as potential hosts for Cryptosporidium. A higher prevalence of Cryptosporidium spp. was observed in larger fish, in fish collected during the spring-summer period, and in those caught in the North East Atlantic. Pollachius virens (saithe) was the most frequently Cryptosporidium positive species. In fish infected by other parasites, the risk of being Cryptosporidium positive increased 10-fold (OR: 9.95, CI: 2.32–40.01.04, P = 0.0002). Four gp60 subtypes were detected among the C. parvum positive samples: IIaA13G1R1, IIaA15G2R1, IIaA17G2R1, and IIaA18G3R1. These C. parvum subtypes have been previously detected in terrestrial mammals and may constitute an additional source of infection for other animals and in particular for humans. Microscopical examination of histological sections confirmed the presence of round bodies suggestive of the development of C. parvum within digestive glands. We report herein the first epidemiological and molecular data concerning the detection of Cryptosporidium in edible marine fish in European seas surrounding France broadening its host range anduncovering potential novel infection routes.

While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples (n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium-specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments.

A total of eight Giardia species are accepted. These include: Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia), which infects humans and animals, Giardia agilis, Giardia ardeae, Giardia psittaci, Giardia muris, Giardia microti, Giardia peramelis and G. cricetidarum, which infect non-human hosts including amphibians, birds, rodents and marsupials. Giardia duodenalis is a species complex consisting of eight assemblages (A-H), with assemblages A and B the dominant assemblages in humans. Molecular studies to date on the zoonotic potential of Giardia in animals are problematic and are hampered by lack of concordance between loci. Livestock (cattle, sheep, goats and pigs) are predominantly infected with G. duodenalis assemblage E, which has recently been shown to be zoonotic, followed by assemblage A. In cats and dogs, assemblages A, B, C, D and F are commonly reported but relatively few studies have conducted molecular typing of humans and their pets and the results are contradictory with some studies support zoonotic transmission but the majority of studies suggesting separate transmission cycles. Giardia also infects a broad range of wildlife hosts and although much less well studied, host-adapted species as well as G. duodenalis assemblages (A-H) have been identified. Fish and other aquatic wildlife represent a source of infection for humans with Giardia via water contamination and/or consumption of undercooked fish and interestingly, assemblage B and A predominated in the two molecular studies conducted to date. Our current knowledge of the transmission dynamics of Giardia is still poor and the development of more discriminatory typing tools such as whole genome sequencing (WGS) of Giardia isolates is therefore essential.

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.

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 Illuminaʼs 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.

Recently, two novel species of Anaplasmataceae were detected in the Australian paralysis tick, Ixodes holocyclus, by 16S rRNA gene metabarcoding. Analysis of these sequences suggested that these novel organisms are closely related to the genus 'Candidatus Neoehrlichia'. In this study, phylogenetic analysis of 16S rRNA (1264 bp), groESL (1047 bp) and gltA (561 bp) gene sequences, and concatenated (2872 bp) sequences, all concur that these novel species belong in the genus 'Candidatus Neoehrlichia' and are most closely related to, but distinct from the only other recognised members of this genus, 'Candidatus Neoehrlichia mikurensis' and 'Candidatus Neoehrlichia lotoris'. Based on their unique molecular signature, we propose to designate these species 'Candidatus Neoehrlichia australis' (reference strain HT41R) and 'Candidatus Neoehrlichia arcana' (reference strain HT94R). Identical 'Candidatus Neoehrlichia australis' 16S rRNA, groESL and gltA sequences were detected in 34/391 (8.7 %) individual Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (96.2 %, 83.1 % and 67.2 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.2 %, 84 % and 68.4 % respectively). Likewise, identical 'Candidatus Neoehrlichia arcana' 16S rRNA, groESL and gltA sequences were detected in 12/391 (3.1 %) Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (98.5 %, 88.7 % and 79.3 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.3 %, 84 % and 67.4 % respectively). These new species are the first Anaplasmataceae (except Wolbachia spp.) to be found to be endemic to Australia. The pathogenic consequences of these organisms are yet to be determined.

Little is known about the epidemiology of Cryptosporidium in Jordan and to date, only one genotyping study has been conducted on Cryptosporidium isolates from Jordanian children. In the present study, a total of 284 faecal samples from Jordanian cattle, sheep, goats and chicken and 48 human faecal samples were screened for the presence of Cryptosporidium using an 18S quantitative PCR (qPCR) and a C. parvum/C. hominis specific qPCR at a lectin locus. Of these, 37 of 284 animal faecal samples were positive by qPCR at the 18S locus giving an overall prevalence of 11.6%. The point prevalence of Cryptosporidium in chickens, sheep, horses, cattle and goats ranged from 4.8% (chickens) to 18.7% (cattle). A total of six species were detected; C. xiaoi (n=9),C. andersoni (n=7),C. ryanae (n=5),C. parvum (n=4),C. baileyi (n=1) and a genetically distinct and potentially novel species in two isolates from horses. Sub-genotype analysis of the 4 C. parvum isolates at the 60-kDa glycoprotein (gp60) locus identified subtype IIaA19G2R1 (n=2) and IIaA16GR1 (n=2). For the human samples, 4 positives (8.3% prevalence) were detected. Of these, two were C. parvum (subtypes IIdA20G1 and IIaA15G2R1) and two were C. hominis (subtypes 1bA9G3 and 1bA10G2R2). Further studies are required to better understand the epidemiology and transmission of Cryptosporidium in Jordan.