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Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oocysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O’Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O’Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O’Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O’Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O’Callaghan & O’Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.

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.

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.

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.

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.

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 (

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.

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.

Objectives To develop molecular tools for the investigation of the prevalence, species and faecal shedding of Yersinia in sheep. Methods A quantitative PCR (qPCR) targeting the β subunit of the Yersinia spp. RNA polymerase gene was developed and validated. The prevalence of pathogenic Y. enterocolitica was determined by screening for the virulent yst gene. These qPCR assays were used to determine Yersinia spp. prevalence and faecal shedding concentration from 3412 faecal samples collected from approximately 1189 lambs (100–180 lambs/flock) on eight farms across Australia. This was a longitudinal study, with sheep sampled on three occasions (weaning, post-weaning and pre-slaughter). A subset of up to five positive samples from each sampling on each farm (n = 111) was sequenced. Results Yersinia spp. (including both pathogenic and non-pathogenic species) were identified in all flocks, with 60.7% of lambs shedding Yersinia spp. on at least one sampling occasion. Point prevalence ranged from 4% to 91% across farms and sampling occasions. Median Yersinia spp. bacterial concentration was 1.1 × 106, 2.8 × 106 and 5.6 × 105 organisms/g faeces at weaning, post-weaning and pre-slaughter, respectively, across all farms. Pathogenic Y. enterocolitica was identified in all eight flocks sampled, with 14.8% of lambs shedding pathogenic Y. enterocolitica on at least one sampling occasion. Conclusion Yersinia spp. and pathogenic Y. enterocolitica in particular were commonly identified in a sample of Australian sheep flocks using molecular techniques. Further studies into associations between faecal shedding of pathogenic Yersinia spp. and sheep productivity or clinical disease may utilise qPCR in conjunction with other diagnostic tools.

A new species, Choleoeimeria pogonae n. sp. is described from a Western bearded dragon (Pogona minor minor) in Western Australia. Sporulated oocysts (n = 48) were cylindroidal in shape. Oocyst length, 27.0 (26.0-28.3) μm, oocyst width, 15.2 (14.0-16.5) μm, oocyst length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts (Eimeria-like) and a polar granule, but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, sporocyst length, 10.0 (9.0-11.0) μm, sporocyst width 8.5 (7.0-9.5) μm, sporocyst L/W ratio, 1.2 (1.1-1.3). Stieda, substieda and parasubstieda bodies were all absent. Molecular analysis was conducted at the 18S rRNA and cytochrome c oxidase I (COI) loci. Phylogenetic analysis of 18S sequences revealed that C. pogonae n. sp. grouped together with another four Choleoeimeria spp. and exhibited 99.1%-99.4% genetic similarity. At the COI locus, C. pogonae n. sp. was in an independent clade and had the highest similarity (80.4%) to Eimeria cf. mivati from a chicken (Gallus gallus domesticus). According to the morphological and molecular data, this isolate is a new species of coccidian parasite. This study further supports the taxonomy of Choleoeimeria spp. as a new genus based on molecular phylogenetic analysis.

This study investigated faecal carriage and antimicrobial resistance (AMR) of Salmonella enterica recovered from rangeland goats. Faecal samples (n = 400) were collected at slaughter from four consignments of goats (n = 100 samples per consignment), each from one of four localities in Western Australia. Carriage of Salmonella spp. was detected in 106 samples (26.5%; 95% CI 22.4–31.0%). The rate of faecal carriage for each consignment ranged between 23–30%. PCR assays targeting the STM2755 and STM4497 genes revealed 84.9% (90/106) of the isolates were of serovar Typhimurium. Salmonella Chester (11/106, 10.4%) and S. Saintpaul (5/106, 4.7%) were characterised at invA and ompF genes. Antimicrobial susceptibility testing demonstrated that 84.0% of isolates were susceptible to all tested (n = 13) antimicrobials. Resistance was identified to azithromycin (14.2%), tetracycline (10.4%), ampicillin (5.7%), amoxicillin–clavulanate and cefoxitin (3.8%), trimethoprim/sulfamethoxazole (1.9%), gentamicin and streptomycin (0.9%). No isolate was resistant to four or more antimicrobials, or to critically important antimicrobials such as fluoroquinolones and extended spectrum cephalosporins. This is the first study reporting AMR in Salmonella isolates from Australian rangeland goats. The rate of detection of AMR was very low, some resistance to low-importance drugs was present in the Salmonella population, despite the absence of active selection pressure.

Cryptosporidium and Giardia are common parasites of ruminant livestock worldwide. These parasites are associated with diarrhoea outbreaks in young goats (pre-weaning), but the impacts on health and productivity for older goats (post-weaning) are not well understood. Here we show Cryptosporidium faecal shedding is associated with reduced growth and diarrhoea in goats aged approximately 9-15 months. Goats were sampled four times at one-month intervals. Faecal shedding for a range of pathogens were determined using quantitative PCR and sequencing (Cryptosporidium, Giardia, Eimeria, Salmonella, Campylobacter), and microscopy (trichostrongylid nematode worm egg count and Entamoeba). Cryptosporidium faecal shedding was associated with 1.5 kg lower growth for the one-month period following sampling. Specifically, C. xiaoi was associated with 1.9 kg lower growth in the following month. This is the first report of production impacts associated with C. xiaoi in ruminants older than 3 months of age. Cryptosporidium shedding was associated with an over 4-fold increase in risk of diarrhoea, with C. parvum associated with 10-fold and C. ubiquitum associated with 16-fold increase in risk of diarrhoea. Notably, C. xiaoi shedding was not associated with increased risk of diarrhoea. Giardia shedding was associated with looser faecal consistency, but not diarrhoea. Higher Eimeria oocyst counts were weakly associated with lower live weight, poorer body condition and looser faecal consistency. Shedding of other enteric pathogens were not associated with impacts on live weight, growth or diarrhoea risk. This study challenges the two notions that Cryptosporidium infections only impact health and productivity of goats during the pre-weaning period, and that Cryptosporidium (and specifically C. xiaoi) infections in the absence of diarrhoea are asymptomatic. Recognising the potential for impacts of Cryptosporidium infection on growth rates in the absence of diarrhoea will support improved design for experiments testing impacts of Cryptosporidium on ruminant health and production. Improved understanding of the role of protozoan infections on animal health has implications for the management of goats in order to reduce adverse impacts on farm profitability, animal welfare and public health risk.

The prevalence of Cryptosporidium and Giardia in faeces collected from sheep at sale yards in Western Australia and for abattoir effluent was determined using a quantitative multiplex PCR (qPCR). A total of 474 faecal samples were collected from sheep at two saleyards on four occasions (April-July 2014) and 96 effluent samples were collected from an abattoir over a four month period (April-July). The overall prevalence of Cryptosporidium in sheep faeces was 6.5% (31/474), with the zoonotic species Cryptosporidium parvum and Cryptosporidium ubiquitum accounting for 54.2% of the typed positive samples. Subtyping of the C. parvum and C. ubiquitum positives at the gp60 locus identified four C. parvum positives as IIdA18G1 and nine C. ubiquitum isolates as the XIId subtype. The overall prevalence of Giardia in sheep faeces was 6.3% (30/474), with the non-zoonotic assemblage E responsible for the majority (81.5%) of positive isolates typed. Median Cryptosporidium and Giardia oo/cyst concentrations in positive faecal samples were 1.7×103 oocysts g-1 (range 32-3.7×106 oocysts g-1) and 2.5×103 cysts g-1 (range 143-7.5×105 cysts g-1) respectively. Cryptosporidium and Giardia were identified in 10.4% (10/96) and 5.2% (5/96) of abattoir effluent samples (respectively). Median Cryptosporidium and Giardia oo/cyst concentrations in positive effluent samples was 1.3×103 cysts g-1 (range 393-1.5×104) and 1.5×104 oocysts g-1 (range 759-4.8×103) respectively. These findings have important implications for the sheep meat industry because Cryptosporidium and Giardia have both been associated with reduced carcase productivity in sheep, and the contamination of lamb carcases and watersheds with zoonotic species have important public health consequences.

A new species, Eimeria collieie n. sp., is described from the western long-necked turtle (Chelodina colliei). Sporulated oocysts (n = 35) are spherical to subspherical, with colourless single layer oocyst wall, 0.6 ± 0.2 (0.4–0.7) µm thick. Oocyst with elongated ellipsoid sporocysts. Oocyst length, 29.8 ± 0.4 (28.2–31.0) µm; oocyst width, 29.4 ± 0.3 (28.0–30.8) µm; oocyst length/width (L/W) ratio, 1.0 ± 0.03 (1.0–1.05). Micropyle, oocyst residuum and polar granule were absent. Sporocysts with sporocyst residuum and 2 sporozoites. Sporocyst length, 21.6 ± 0.4 (21.2–22.0) µm; sporocyst width, 6.0 ± 0.3 (5.7–6.3) µm; sporocyst L/W ratio, 3.6 ± 0.2 (3.4–3.8). Stieda, parastieda and substieda bodies were absent. Sporozoite length, 14.0 ± 0.2 (13.8–14.2) µm; sporozoite width, 2.6 ± 0.2 (2.4–2.8) µm; sporozoite L/W ratio, 5.46 ± 0.10 (5.4–5.6). Molecular analysis was conducted at three loci: the 18S and 28S ribosomal RNA (rRNA), and the mitochondrial cytochrome oxidase gene (COI). At the 18S rRNA locus, E. collieie n. sp. shared 96.4% and 98.3% genetic similarity to E. ranae (GenBank accession number: EU717219) and E. arnyi (AY613853) respectively. At the 28S rRNA locus, E. collieie n. sp. shared 91.6% genetic similarity to E. papillata (GenBank accession number: GU593706) and phylogenetic analysis at this locus placed E. collieie n. sp. in aseparateclade. At the COI locus, E. collieie n. sp. shared 92.7% genetic similarity to Eimeria setonicis (GenBankaccession number: KF225638) from a quokka (Setonix brachyurus) in Western Australia. Reptile-derived sequences were not available for the 28S rRNA and the COI loci. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that, to date, has only been found in western long-necked turtles.

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-basedgenotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the “true” composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled.

A new species, Isospora streperae n. sp., (Apicomplexa: Eimeriidae) is described from a single grey currawong bird (Strepera versicolour) (subspecies S. v. plumbea) in Western Australia. Sporulated oocysts (n = 32) are spherical to subspherical, with smooth colourless bilayered oocyst wall, 1.0 μm thick (outer layer 0{dot operator}8 μm, inner 0.2 μm thick). Oocyst with a polar granule, an oocyst residuum and two spheroidal to subspheroidal sporocysts. Oocyst length, 23.8 (20.4-25.0) μm; oocyst width, 22.5 (20.0-24.6) μm; a shape index of 1.06, with Stieda, substieda bodies. Micropyle is absent. Sporocysts with compressed sporocyst residuum and four sporozoites. Sporocyst length, 14.4 (12.5-15.2) μm; sporocyst width, 11.2 (10.6-14.0) μm, sporocyst L/W ratio, 1.29. Necropsy of the bird identified haemorrhaging along the ileum and jejunum, which is where Isospora oocysts were also mostly detected. Molecular analysis was conducted at three loci; the 18S, 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, I. streperae n. sp. exhibited 99.5% and 99.4% similarity respectively to an Isospora sp. (MS-2003) from a Southern cape sparrow (Passer melanurus melanurus) and Isospora dovati from a domestic pigeon (Columba livia domestica). At the 28S locus, I. streperae n. sp. exhibited 96.9% similarity to an Isospora sp. (MS-2003) from a grosbeak starling (Scissirostrum dubium) and 95.8% similarity with the Isospora sp. (MS-2003) from a Southern cape sparrow. At the COI locus, I. streperae n. sp. exhibited 95.0% similarity to Isospora sp. from a yellow-necked mouse (Apodemus flavicollis) from the Czech Republic. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora streperae n. sp. after its host, the grey currawong (Strepera versicolour plumbea).

Faecal excretion of Campylobacter spp. and Salmonella enterica in sheep in Australia was determined using a quantitative multiplex PCR (qPCR) targeting the Campylobacter spp. purine biosynthesis gene (PurA) and the S. enterica outer membrane protein (ompF). The mutiplex qPCR was specific and Campylobacter spp. and S. enterica were each detected with a sensitivity of 5 organisms/µL faecal DNA extract. This multiplex qPCR was used to determine the prevalence and concentration of Campylobacter spp. and S. enterica in 3412 faecal samples collected from 1189 lambs on eight farms across South Australia (n = 2 farms), New South Wales (n = 1), Victoria (n = 2) and Western Australia (n = 3) at three sampling periods (weaning, post-weaning and pre-slaughter). The overall prevalences of Campylobacter spp. and S. enterica were 13.3% and 5.0%, respectively, with the highest prevalence for Campylobacter spp. in South Australia and the highest prevalence for S. enterica in New South Wales. Campylobacter jejuni was the only Campylobacter sp. identified from a subset of 120 positive samples sequenced at the 16S locus. S. enterica serovar Typhimurium was the only serovar of S. enterica identified from a subset of 120 positive samples sequenced at the ompF locus. Across all states, Campylobacter spp. had the highest median bacterial concentration in faeces at weaning and post-weaning (medians of 3.4 × 10(6) and 1.1 × 10(5), respectively), whereas S. enterica had the highest median bacterial concentration at pre-slaughter (1.8 × 10(5)/g faeces).

The prevalence of Cryptosporidium in sheep in the eastern states of Australia has not been well described, therefore a study of the prevalence, oocyst concentration, species and subtypes of Cryptosporidium were assessed from lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected from approximately 1182 lambs across the four states and screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) at the actin locus. Positives were typed at the 18S locus and at a second locus using C. parvum and C. hominis specific qPCR primers. The overall prevalence was 16.9% (95% CI: 15.6–18.1%) and of the 576 positives, 500 were successfully genotyped. In general, the prevalence of Cryptosporidium was higher in WA than the eastern states. Cryptosporidium prevalence peaked at 43.9% and 37.1% at Pingelly (WA2) and Arthur River (WA1), respectively during weaning and at Pingelly (WA2) during pre-slaughter (36.4%). The range of oocyst shedding at weaning overall across all states was 63–7.9 × 106 and the median was 3.2 × 104 oocysts g−1. The following species were identified; C. xiaoi (69%—345/500), C. ubiquitum (17.6%—88/500), C. parvum (9.8%—49/500), C. scrofarum (0.8%—4/500), mixed C. parvum and C. xiaoi (2.4%—12/500), C. andersoni (0.2%—1/500) and sheep genotype 1 (0.2%—1/500). Subtyping of C. parvum and C. ubiquitum isolates identified IIa and IId subtype families within C. parvum (with IId as the dominant subtype) and XIIa within C. ubiquitum. This is the first published description of C. parvum subtypes detected in lambs in Australia.

A novel quantitative PCR (qPCR) for Giardia at the glutamate dehydrogenase (gdh) locus was developed and validated. The qPCR was used to screen a total of 3412 lamb faecal samples collected from approximately 1189 lambs at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia (SA), New South Wales (NSW), Victoria (Vic) and Western Australia (WA). The overall prevalence was 20.2% (95% CI 18.9-21.6) and of the 690 positives, 473 were successfully typed. In general, the prevalence of Giardia varied widely across the different farms with the highest prevalence in one WA farm (42.1%) at pre-slaughter sampling and the lowest prevalence in one Victorian farm (7.2%) at weaning. The range of cyst shedding at weaning, post-weaning and pre-slaughter overall across all states was 63-1.3×10(9) cysts g(-1) (median=1.7×10(4)), 63-1.1×10(9) cysts g(-1) (median=9.6×10(3)), 63-4.7×10(9) cysts g(-1) (median=8.1×10(4)) respectively. Assemblage specific primers at the triose phosphate isomerase (tpi) locus identified assemblage A in 22.4% (106/473) of positive samples typed, assemblage E in 75.9% (359/473) and mixed A and E assemblages in 1.7% (8/473) of samples. A subset of representative samples from the 8 farms (n=32) were typed at both the gdh and beta-giardin loci and confirmed these results and identified sub-assemblage AII in 16 representative assemblage A isolates across the 8 farms. This demonstrates a prevalence of Giardia previously not recognised in Australian sheep, highlighting a need for further research to quantify the production impacts of this protozoan parasite.

A new species, Eimeria tiliquae n. sp. is described from a shingleback skink (Tiliqua rugosa rugosa). Sporulated oocysts (n=. 50) are spherical to subspherical, with colourless trilaminate oocyst wall, 0.7 ± 0.1 (0.5-0.75) thick. Oocyst with 4 spheroidal to subspheroidal sporocysts. Oocyst length, 13.7 ± 0.9 (12.0-16.3); oocyst width, 12.8 ± 0.9 (11.5-15.0); oocyst length/width (L/W) ratio, 1.07 ± 0.05 (1.0-1.2). Micropyle, oocyst residuum and polar granule absent. Sporocysts with globular sporocyst residuum and 2 sporozoites. Sporocyst length, 6.0 ± 0.6 (5.0-7.5); sporocyst width, 5.4 ± 0.6 (4.0-7.0); sporocyst L/W ratio, 1.11 ± 0.11 (1.0-1.5). Stieda, parastieda and substieda bodies absent. Phylogenetic analysis of 18S rRNA sequences indicated that E. tiliquae n. sp. shared 96.4-96.5% genetic similarity to E. tropidura, its closest relative. Reptile-derived sequences were not available for the mitochondrial cytochrome oxidase gene (COI) and phylogenetic analysis at this locus placed E. tiliquae n. sp. in a clade by itself but grouping closest (92% similarity) with a novel isolate from a King's skink (Egernia kingii) from Western Australia. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in shingleback skinks.

Faecal shedding of Cryptosporidium and Giardia by captured rangeland goats was investigated using a longitudinal study with four faecal samples collected from 125 male goats once monthly for four months, commencing immediately after capture and transport to a commercial goat depot (feedlot). Goats were composite breed and aged approximately 9-12months on arrival. Faecal samples were screened for Cryptosporidium and Giardia presence and concentration using quantitative PCR and sequencing at the 18S ribosomal RNA locus (Cryptosporidium), and glutamate dehydrogenase and β-giardin loci (Giardia). Longitudinal prevalence for Cryptosporidium was 27.2% (point prevalence range 3-14%) with 3 species identified: C. xiaoi (longitudinal prevalence 13.6%), C. ubiquitum (6.4%) and C. parvum (3.2%). Sub-typing at the gp60 locus identified C. ubiquitum XIIa, C. parvum IIaA17G2R1 and C. parvum IIaA17G4R1. This is the first report of the zoonotic C. parvum subtype IIaA17G4R1 in goats. The pattern of genotypes shed in faeces changed over the duration of study with C. ubiquitum identified only at the first and second samplings, and C. parvum identified only at the fourth sampling. Longitudinal prevalence for Giardia duodenalis was 29.6% (point prevalence range 4-12%) with all positives sub-typed as assemblage E. Only 2/125 goats were identified to be shedding Cryptosporidium or Giardia on more than one occasion. This is the first report of Cryptosporidium and Giardia genotypes in captured rangeland goats. Faecal shedding of zoonotic Cryptosporidium spp. and potentially zoonotic G.duodenalis has implications for food safety and effluent management. Keywords: Cryptosporidium; Giardia; Rangeland goats; zoonotic.

Background Cystoisosporiasis is an opportunistic infection seen more commonly in patients with acquired immunodeficiency syndrome. Although uncommon, Cystoisospora infection can occur in immunocompetent individuals but tend to be benign and self-limiting. Chronic infection however, has been described but diagnosis can often be challenging and requires a high clinical index of suspicion. Case presentation We present a case of delayed diagnosis of Cystoisospora belli (C. belli) in an immunocompetent 28-year-old refugee from Myanmar. She had a history of chronic diarrhea where exhaustive investigations over many years failed to reveal a diagnosis. Cystoisospora belli cysts were finally detected in stool 4 years after investigation commenced, and PCR testing on stored colon biopsies amplified a molecular product with 99 % sequence homology to C. belli. The patient improved promptly with trimethoprim-sulfamethoxazole treatment. Conclusion In the appropriate clinical context we suggest molecular testing for C. belli or an empirical therapeutic trial.

Parasites of wildlife inhabiting urbanised and peri-urban environments are of interest regarding wildlife population health, and also veterinary public health in the case of parasites that can also infect humans and domestic animals. This study aimed to: identify, and estimate the prevalence of, species of Eimeria parasitic in quenda (Isoodon obesulus) in the greater Perth region, Western Australia; 2) morphologically describe and genetically characterise a novel observed species of Eimeria as E. angustus; and 3) genetically characterise E. kanyana. Eimeria spp. prevalence was 76.1% (95% CI 64.9–84.5%), and four putative species of Eimeria were identified. Eimeria kanyana was identified infecting quenda for the first time, with a prevalence of 54.9% (43.4–66.0%). Eimeria quenda was less prevalent, at 7.0% (3.1–15.5%). The novel species E. angustus was present in 45.1% of sampled quenda (34.0–56.6%). A second novel morphotype of Eimeria was present in 2.8% of sampled quenda (0.9–9.7%). Mixed Eimeria spp. infections were present in 21/71 quenda (29.6%, 95% CI 20.2–41.1%). Molecular phylogenetic analyses of E. kanyana and E. angustus were conducted at the 18S rRNA and mitochondrial cytochrome oxidase loci. At both loci, two isolates identified as E. kanyana grouped in a phylogenetic clade with E. trichosuri. Five isolates identified as the novel E. angustus were most closely related to E. tropidura at the 18S locus. At the COI locus, no sequence data were available for E. tropidura; isolates of E. angustus grouped with E. sciurorum.

Little is known about the epidemiology of Giardia in Jordan and to date, no genotyping studies have been conducted on Giardia isolates from Jordanians. In the present study, a total of 49 microscopy-positive faecal samples from Jordanian patients suffering from giardiasis were analysed at two loci: the triose phosphate isomerase (tpi) gene and the glutamate dehydrogenase (gdh) gene. At the tpi locus, a total of 28 samples amplified and assemblage A was identified in 46.4 % (13/28) samples, while assemblage B was identified in 50 % (14/28) samples and a mixed assemblage A and B was identified in one sample (3.6 %) (Table 1). At the gdh locus 48 isolates amplified and of these assemblages A was identified in 43.7 % (21/48) of isolates and assemblage B in 56.3 % (27/48) of isolates. No mixed infections were detected at the gdh locus. Subtyping at the gdh locus identified sub-assemblage AII in 43.7 % (21/48) of isolates and sub-assemblages BIII and BIV in 25 % (12/48) and 31.2 % (15/48) of isolates, respectively, with more genetic diversity in AII isolates than BIII or BIV isolates. Novel sub-types within each sub-assemblage were identified suggesting unique endemicity and anthroponotic transmission of Giardia in Jordanian patients suffering from giardiasis. Further studies are required to better understand the epidemiology and transmission of Giardia in Jordan.

Associations between intensity and frequency of Cryptosporidium and Giardia shedding with growth, carcase weight and dressing% were investigated using a longitudinal study of 1182 lambs on eight Australian farms. Live weight was recorded and faecal samples were collected on three sampling occasions; weaning (approximately 12 weeks of age), post-weaning (approximately 19 weeks) and pre-slaughter (approximately 29 weeks). Hot standard carcase weight (HSCW) and dressing% were measured at slaughter. Faecal samples were screened for presence and concentration of Cryptosporidium, Giardia and Haemonchus oocysts using a quantitative PCR. Trichostrongylid eggs were quantified with modified McMaster faecal worm egg count (WEC). Protozoan shedding intensity was categorised as high (above median oocyst concentration in positive sheep), low (below median oocyst concentration in positive sheep) or not detected. Shedding was also categorised for shedding type (no shedding, single Giardia infection, single Cryptosporidium infection, concurrent Giardia and Cryptosporidium infection) and lambs were categorised for frequency of shedding (shedding identified on 0, 1, 2 or 3 occasions). Associations of parasite shedding intensity category, shedding type, shedding frequency, WEC and Haemonchus status (positive or negative) with lamb production were assessed using general linear models (HSCW and dressing%) and linear mixed effects models (live weight). High Cryptosporidium parvum shedding was associated with lower live weight, ranging 2.31–4.52 kg over the 3 sampling occasions. Cryptosporidium parvum shedding was associated with less HSCW in high (3.22 kg less) and low (3.22 kg less) shedding lambs post-weaning, and high (2.21 kg less) and low (2.60 kg less) shedding lambs pre-slaughter as well as lower dressing% (2.7% lower in high shedding lambs post-weaning). Cryptosporidium (all species) shedding pre-slaughter was associated with reduced dressing% in both high (1.25% lower) and low (1.21% lower) shedding lambs. Giardia shedding pre-slaughter was associated with 0.59 kg less HSCW in high shedding lambs. Increased frequency of C. parvum and Giardia shedding in a specific animal (repeated detection) were associated with reduced HSCW and dressing%. Concurrent Giardia and Cryptosporidium shedding pre-slaughter was associated with reduced dressing%. No statistically significant main effects for either WEC (P > 0.05) or Haemonchus status (P > 0.05) were identified for any of the sheep meat productivity measures (live weight, HSCW and dressing%). The findings suggest naturally acquired Cryptosporidium and Giardia infections in grazing sheep are associated with depressed growth, carcase weight and dressing efficiency beyond the neonatal period in sheep representing a range of genetic backgrounds and different sheep production environments.

usc Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney's drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5' half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3' end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may have important implications for the management of drinking water catchments to minimize risk to public health. © 2016 Zahedi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Refereed/Peer-reviewed

We investigated the role of the western grey kangaroo (Macropus fuliginosus) in the maintenance and transmission of Coxiella burnetii in Western Australia. Sera from 1,017 kangaroos were tested using an indirect enzyme-linked immunosorbent assay (ELISA) for the presence of C. burnetii antibodies. The overall antibody prevalence across 12 locations throughout mid- to southwestern Western Australia was 24.1% (95% CI: 21.6-26.8). Feces from 990 of the same animals were tested using PCR to identify active shedding of C. burnetii in excreta. Coxiella burnetii DNA was detected in 4.1% (95% CI: 3.1-5.6) of samples. Our results suggest that kangaroos are reservoirs for C. burnetii in Western Australia and may contribute to transmission of the organism to domestic livestock and humans.

A total of 477 faecal samples from pre-weaned sheep from 5 different farms in the south west of Western Australia were screened for the presence of Cryptosporidium and Giardia using PCR. There were substantial differences in prevalence between the farms and overall prevalence was 24.5% and 11.1%, respectively for Cryptosporidium and Giardia. At the 18S locus, 66 Cryptosporidium positives were identified, the majority of which were C. bovis (n = 52), followed by the cervid genotype (n = 10) and C. parvum (n = 2). At a second diagnostic locus, using C. parvum and C. hominis-specific qPCR primers, 63 C. parvum positives were identified, some of which were co-infections with C. bovis. The C. parvum/C. hominis qPCR was more sensitive than the nested 18S PCR at detecting C. parvum. This may be due to the low numbers of oocysts present, as quantitation data indicated that all the C. parvum detected were present in low numbers (1–10 oocysts). It may also be that using C. parvum-specific primers is necessary to determine the true prevalence of C. parvum. Amongst Giardia positive isolates, G. duodenalis genotype E (livestock) was the most prevalent (36/53), with G. duodenalis genotype A detected in five positive isolates. There were also 11 mixed A and E infections detected. The findings of the present study indicate that pre-weaned lambs are not an important source of zoonotic Giardia genotypes in Australia but may be an important source of zoonotic Cryptosporidium.

A total of 421 fecal samples from a variety of captive and wild marsupial hosts in Western Australia, Victoria and South Australia were screened for the presence of Giardia species/genotypes using PCR and sequence analysis of a fragment of the 18S rRNA gene. Giardia spp. were identified in 13.4% (28/209) of samples from captive marsupials and 13.7% (29/212) of samples from wild marsupials. Sequence analysis at the 18S locus identified the zoonotic Giardia duodenalis Genotypes A and B in both captive and wild marsupials. Eight isolates were typed as genotype B3 and B4 at the gdh locus, although 7/8 were typed as genotype A at the 18S rRNA locus. The possible reasons for this discordance are discussed. This is the first report of genotype B and only the second report of genotype A in marsupials. As some of the genotype B isolates were identical to human-derived Giardia gdh sequences, these results suggest that marsupials in catchments may pose a public health risk and therefore warrant further investigation.

A new Isospora (Apicomplexa:Eimeriidae) species is described from a single yellow-throated miner bird (Manorina flavigula) (subspecies M. f. wayensis) in Western Australia. Sporulated oocysts (n = 32) of this isolate are spherical to subspherical, 22.8 (20.3-23.8) × 18.3 (17.7-18.7) μm, with a shape index (length/width) of 1.25 (1.2-1.3); and a smooth and bilayered oocyst wall, 1.3 μm thick (outer layer 0.9 μm, inner 0.4 μm). A polar granule is present, but the micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 15.5 (14.6-15.8) × 9.5 (9.5-10.2) μm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being knob-like and the substieda body being subspherical-shaped. A sporocyst residuum is present and composed of numerous granules of different size scattered among the sporozoites, a spheroid or subspheroid refractile body is present in the sporozoite. Morphologically, the oocysts from this isolate are different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci; the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, this new isolate exhibited 99.2% similarity to Isospora gryphoni and three other Isospora spp. Further analysis of a subgroup of 300 bp long 18S sequences (8), including Isospora anthochaerae was conducted. This new isolate grouped in a clade with I. anthochaerae and exhibited 99.3% similarity. At the 28S locus, this new isolate grouped with I. anthochaerae with which it shared 99.1% similarity. At the COI locus, this new isolate exhibited 96.8% similarity to Isospora sp. JCI-2015 from a spectacled warbler (Sylvia conspicillata) in Spain. Further analysis from a subgroup of shorter COI sequences (n = 13) was performed and this new isolate exhibited 99.1% similarity to I. anthochaerae. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora manorinae n. sp. after its host, the yellow-throated miner (Manorina flavigula wayensis).

A new species, Isospora serinuse n. sp., (Apicomplexa:Eimeriidae) is described from a single domestic canary (Serinus canaria forma domestica) (subspecies S. c. domestica) in Western Australia. Sporulated oocysts of Isospora serinuse n. sp. are spherical or subspherical, 25.5 (24.4-27.0) × 23.5 (22.0-24.8) μm, with a shape index (length/width) of 1.09; and a smooth bilayered oocyst wall, 1.2 μm thick (outer layer 0.9 μm, inner 0.3 μm). A polar granule is present, but a micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 18.9 (17.8-20.2) × 11.8 (10.6-13.0) μm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being a small crescent shape and the substieda being indistinct. Each sporocyst with four vermiform sporozoites arranged head to tail. A sporocyst residuum is present and composed of numerous granules of different sizes that are scattered among the sporozoites. Morphologically, the oocysts of Isospora serinuse n. sp. were different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci: the 18S and 28S ribosomal RNA and two separate regions of subunit I of the mitochondrial cytochrome oxidase (COI) gene (designated COIa and COIb). At the 18S locus, Isospora serinuse n. sp. exhibited 97.5% similarity to Isospora sp. Tokyo from a domestic pigeon (Columba livia domestica) in Japan. At the 28S locus, I. serinuse n. sp. exhibited 94.9% similarity to Isospora anthochaerae n. sp. from a red wattlebird (Anthochaera carunculata) in Australia. At the COIa locus, I. serinuse n. sp. exhibited 95.7% similarity to Isospora sospora sp. ex Apodemus flavicollis from a yellow-necked mouse and Isospora gryphoni from an American goldfinch (Carduelis tristis) respectively. At the COIb locus, I. serinuse n. sp. exhibited 96.7% similarity to an Isospora (iSAT4) from a European pied flycatcher (Ficedula hypoleuca). Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora serinuse n. sp. after its host, the domestic canary (S. canaria forma domestica).

Background: Despite a long history of safe use and the incorporation of probiotics into current therapeutic guidelines, the ability of yogurt and its probiotics to colonize the gastrointestinal tract, the appropriate mode of administration in order to maximize probiotic efficacy remain uncertain. Aim: To determine the effect of daily probiotic supplementation of Lactobacillus acidophilus La5 (La5) and Bifidobacterium animalis subsp lactis BB12 (BB12) from capsules and the whole food (yogurt) form, on faecal counts of La5, BB12, and total Bifidobacteria. Methods: Following a 3-week washout period, 156 men and women over 55 y were randomized to a 6-week double-blinded parallel study. The four intervention groups were: A) probiotic yogurt plus probiotic capsules; B) probiotic yogurt plus placebo capsules; C) control milk plus probiotic capsules; and D) control milk plus placebo capsules. Results are presented as the median (IQR) number of bacteria per g feces. Results: When compared to control milk, probioti...

Two hundred and forty calf faecal samples from 16 Malaysian farms were screened by PCR for Giardia spp. The overall prevalence was 12.5% and the overall farm prevalence was 68.8% (11/16 farms). The prevalence in pre-weaned and weaned calves was 16.7% and 8.3%, respectively. Sequence analysis of 25 isolates identified all as G. duodenalis assemblage E. Management factors associated with an increased risk of infection with Giardia spp. included keeping weaned calves in pens with sand floors and calf age. Keeping pre-weaned calves in pens with concrete floors and calving in single cow calving areas decreased the risk.

Little is known of the prevalence of Cryptosporidium and Giardia in domestic cats in Western Australia and their potential role as zoonotic reservoirs for human infection. In the present study, a total of 345 faecal samples from four different sources were screened for the presence of Cryptosporidium and Giardia by PCR and genotyped by sequence analysis. Oocyst numbers and cyst numbers for Cryptosporidium and Giardia respectively were also determined using quantitative PCR assays. Cryptosporidium and Giardia were detected in 9.9% (95% CI 6.7-13.0) and 10.1% (95% CI 7.0-13.3) of cats in Western Australia respectively. Sequence analysis at the 18S rRNA locus identified five Cryptosporidium species/genotypes; C. felis (n = 8), C. muris (n = 1), C. ryanae (n = 1), Cryptosporidium rat genotype III (n = 5) and a novel genotype most closely related to Cryptosporidium rat genotype III in one isolate. This is the first report of C. ryanae and Cryptosporidium rat genotype III in cats. For Giardia, assemblage F the most commonly identified species, while only 1 assemblage sequence was detected. Since most human cases of cryptosporidiosis are caused by C. parvum and C. hominis and human cases of giardiasis are caused by G. duodenalis assemblage A and B, the domestic cats in the present study are likely to be of low zoonotic risk to pet owners in Perth. Risk analyses identified that elderly cats (more than 6 years) were more prone to Cryptosporidium and Giardia infections than kittens (less than 6 months) (P = 0.009). Clinical symptoms were not associated with the prevalence of Cryptosporidium and Giardia infections in cats. Refereed/Peer-reviewed

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R2≥0.999) and faecal samples (R2≥0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies. Refereed/Peer-reviewed

An Eimeria species is described from a dusky moorhen (Gallinula tenebrosa). Sporulated oocysts (n = 40) are ovoid, with a pitted single-layered oocyst wall in young oocysts and a relatively smooth wall in the mature oocysts. Oocyst wall was 1.0 µm thick, oocysts measured 17.3 × 13.3 (16.3-17.9 × 12.7-13.9) µm, oocyst length/width (L/W) ratio, 1.3. Oocyst residuum was absent. A large polar granule was always observed in the centre of the micropyle and many small polar granules were observed when the focus was on the wall. Sporocysts are elongate-ovoid, 8.4 × 5.1 (8.0-8.9 × 4.9-5.5) µm, sporocyst L/W ratio, 1.6 (1.5-1.8), sporocyst residuum was present, composed of numerous granules in a spherical or ovoid mass. Each sporocyst contained 2 elongate sporozoites, 7.7 × 2.6 (7-10 × 2.2-3) µm. A spherical-ellipsoid posterior refractile body was found in the sporozoites. A nucleus is located immediately anterior to the posterior refractile body. When the oocyst measurements and features were compared with valid Eimeria species from hosts in the Rallidae family, this Eimeria species was identified as E. paludosa. This is the first report of E. paludosa in Australia and the dusky moorhen (Gallinula tenebrosa) in a new host for this species. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the mitochondrial cytochrome oxidase gene (COI). At the 18 S locus, E. paludosa shared 97.3% genetic similarity with Eimeria gruis (GenBank accession number: AB544336). It also shared 99.2% genetic similarity with Eimeria crecis (GenBank accession numbers: HE653904 and HE653905) and 98.5% similarity with Eimeria nenei (GenBank accession numbers: HE653906), both of which were identified from a corncrake (Crex crex) in the United Kingdom. At the 28S locus, E. paludosa shared 91.4% similarity with E. papillata from a chicken (Gallus gallus) in the USA. At COI locus, E. paludosa was in a clade by itself and shared 87.2% similarity with E. irresidua, from a European rabbit (Oryctolagus cuniculus) from the Czech Republic. This is the first molecular characterization of E. paludosa.

A new Caryospora coccidian species is described from the laughing kookaburra (Dacelo novaeguineae). Sporulated oocysts (n=30) are ovoid in shape with a smooth, colourless, bilayered oocyst wall and measure 31.4×29.3 (30.0-32.0×28.0-31.0) μm with a shape index of 1.1. Oocysts contain one spheroidal to subspheroidal sporocyst, 21.2×20.6 (20.0-24.0×20.0-21.0) μm. A spheroidal shaped sporocyst residuum is present; micropyle, Stieda, substieda and parastieda bodies are absent. Vermiform sporozoites (n=8) are arranged either parallel or randomly in the sporocyst, measuring 17.0×4.8 (16.0-18.0×4.0-6.0) μm, with a L/W ratio of 3.5. There is a large spheroidal, posterior refractile body in the middle of the sporozoite. Morphologically, this new species is most similar to Caryospora. The prevalence of this parasite was 6.7% in birds sampled in the morning and 33.3% from those sampled after midday. Further molecular characterisation was conducted at two loci; the 18S and 28S ribosomal RNA (rRNA). At the 18S locus, the new species of Caryospora was most closely related to Besnoitia besnoiti (99.2% similarity) and Hammondia triffittae (98.8% similarity). Although, no 28S partial sequences from Caryospora were available in GenBank, the highest similarity was with B.besnoiti (91.3%). Based on morphological and molecular data, this coccidian parasite is a new species that to date has not been reported. The new coccidian parasite is named Caryospora daceloe n. sp. after its host D. novaeguineae (the laughing kookaburra).

The prevalence and faecal shedding of Chlamydia spp. in sheep in Australia has not been well described. Two species-specific quantitative PCRs (qPCRs) targeting the chlamydial outer membrane protein cell surface antigen gene (ompA) were validated and used to determine the prevalence and faecal shedding of C. abortus and C. pecorum from faecal samples of lambs at three sampling times (weaning, post-weaning and pre-slaughter) from eight farms in South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected and screened from approximately 1189 lambs across the four states. C. abortus was not detected in any of the samples screened. The overall prevalence of C. pecorum was 1027/3412 (30.1%) and median bacterial concentrations at weaning, post-weaning and pre-slaughter were 1.8 × 10(7), 1.2 × 10(7) and 9.6 × 10(5)/g faeces, respectively. A subset of C. pecorum positive samples from each farm, (n = 48) was sequenced to confirm their identity. The present study demonstrates that C. pecorum is prevalent in Australian sheep, highlighting a need for further research on the impact of this bacterium on production.

A new species, Isospora anthochaerae n. sp. is described from a Red wattlebird (Anthochaera carunculata). Sporulated oocysts (n=37) are subspherical, with smooth colourless to pale brown bilayered oocyst wall, 0.8 μm thick (outer layer 0·6 μm, inner 0.2 μm thick). Oocyst with 2 spheroidal to subspheroidal sporocysts. Oocyst length, 23.4 μm (20.0-26.0); oocyst width, 20.7 μm (19.0-22.0); oocyst length/width (L/W) ratio, 1.1. Micropyle, oocyst residuum and polar granule are absent. Sporocysts with compact sporocyst residuum and 4 sporozoites. Sporocyst length, 14.5 μm; sporocyst width, 10.1 μm sporocyst L/W ratio, 1.4. Molecular analysis was conducted at four loci; the ribosomal internal transcribed spacer (ITS), the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase gene (COI). At the COI locus, I. anthochaerae n. sp. exhibited 98.5% similarity to Isospora lesouefi from a Regent honeyeater (Xanthomyza phrygia) and 98% similarity with an Isospora sp. (iSAT5) from a blackcap (Sylvia atricapilla). Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in Red wattlebirds.

The prevalence of Eimeria in sheep in Australia has not been well described, therefore a quantitative PCR (qPCR) was developed, validated and used to study the prevalence and oocyst concentration in lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected from approximately 1182 lambs across the 4 states and screened for the presence of Eimeria using this qPCR at the 18S ribosomal RNA (rRNA) locus. A subset of positives was typed by sequence analysis at the 18S locus. The overall prevalence was 18.1% (95% CI 16.8-19.3%) and of the 616 positives, 118 were successfully genotyped. The prevalence of Eimeria was highest in NSW and peaked at 70% during the post-weaning period. The range of oocyst shedding per gram of faeces (g(-1)) at weaning, post-weaning and pre-slaughter overall across all states was 23-2.1×10(7), 23-1.3×10(7) and 23-2.1×10(5), respectively. Median Eimeria shedding g(-1) was higher during post-weaning (1.1×10(3)) and pre-slaughter (1.1×10(3)) than during weaning (206). The following species were identified: Eimeria crandallis, Eimeria ahsata, Eimeria ovinoidalis, Eimeria weybridgensis and Eimeria cylindrica. Of these, E. crandallis and E. ovinoidalis, the most pathogenic species in sheep were responsible for 58.5% of infections typed. This highlights a need for further research to quantify the production impacts of Eimeria in sheep.

The identification and characterisation of novel Eimeria species has largely been based on sporulated oocyst and sporocyst morphology, the host species and the geographical range. Variation in the size and shape of Eimeria oocysts across their host range however, make the identification and characterisation of novel species using traditional methodologies alone problematic. The use of molecular markers and phylogenetic analysis has greatly advanced our ability to characterise Eimeria species and has recently been applied to understand evolutionary relationships among Eimeria species from Australian marsupials. In the present study, Eimeria species isolated from quokkas (Setonix brachyurus) captured from Two Peoples Bay, Bald Island and Rottnest Island, Western Australia, were morphologically identified as Eimeria quokka and Eimeria setonicis. Both Eimeria species were identified as being polymorphic in nature with regards to sporulated oocyst and sporocyst morphometrics. Phylogenetic analysis using 18S rRNA and COI (cytochrome c oxidase subunit 1) genes, grouped E. quokka and E. setonicis within the Eimeria marsupial clade together with Eimeria trichosuri from brushtail possums, Eimeria macropodis from tammar wallabies (Macropus eugenii) and several unidentified macropod Eimeria species from western grey kangaroos (Macropus fuliginosus). This study is the first to characterise E. quokka and E. setonicis by molecular analysis, enabling more extensive resolution of evolutionary relationships among marsupial-derived Eimeria species.

A total of 597 faecal samples were collected from western grey kangaroos (Macropus fuliginosus), Euros (M. robustus), red kangaroos (M. rufus) in Western Australia and Eastern Grey Kangaroos (M. giganteus) from Victoria and screened for the presence of Eimeria by PCR at the 18S ribosomal RNA (rRNA) locus. The overall prevalence was 24.3% (145/597). At the 18S rRNA locus, sequences were obtained for 25 of the 145 positives. Phylogenetic analysis indicated that all the macropod-derived Eimeria species grouped in a separate marsupial clade that included Eimeria trichosuri from brushtail possums. At least 6 different clades were identified within the marsupial isolates and many of the genotypes identified are likely to be valid species, however morphological and biological data need to be collected to match sequences to previously characterized Eimeria species or identify if they are new species.

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2×10(4) oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.

A total of 763 faecal samples were collected from western grey kangaroos (Macropus fuliginosus) in Western Australia and screened for the presence of Cryptosporidium by PCR at the 18S ribosomal RNA (rRNA) locus. Samples that were positive at the 18S locus were also amplified at the actin locus. The overall prevalence was 9.3% (71/763). At the 18S rRNA locus, sequences were obtained for 28 of the 71 positives. Sequence analysis identified four species; Cryptosporidium fayeri in seven isolates, Cryptosporidium marcopodum in four isolates, Cryptosporidium xiaoi in six isolates and a novel genotype (kangaroo genotype I) in eleven isolates. Analysis at the actin locus confirmed the genetic distinctness of the novel genotype. The results of the present study indicate that in addition to C. fayeri and C. marcopodum, kangaroos may be capable of being infected with a wider range of Cryptosporidium species and genotypes including livestock species such as C. xiaoi. The novel genotype identified in the kangaroos most likely represents a cryptic species that requires further analyses to confirm its species status.

In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-acre, commercial sheep farms in southern Western Australia, on two separate occasions (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old), were individually identified using ear tags (a numbered tag and a radio-frequency tag). A total of 1155 faecal samples were collected only from these individually identified lambs on five separate sampling occasions. All samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). The overall prevalences (lambs positive for a parasite on at least one of the five samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia prevalences at individual samplings ranged between 18.5 and 42.6% in lambs and were