Morphological and molecular characterization of Eimeria haematopusi n. sp. (Apicomplexa: Eimeriidae) in an Australian Pied Oystercatcher (Haematopus longirostris) (Aves: Charadriiformes).

A novel Eimeria Schneider, 1875 species is described from an Australian pied oystercatcher Haematopus longirostris Vieillot, in Western Australia. The pied oystercatcher was admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth, Western Australia in a poor body condition, abrasion to its right hock and signs of partial delamination to its lower beak. Investigation into potential medical causes resulted in a faecal sample being collected and screened for gastrointestinal parasites. Unsporulated coccidian oocysts were initially observed in the faeces and identified as Eimeria upon sporulation. The sporulated oocysts (n = 20) are ellipsoidal, 20-21 × 12-13 µm in shape and have thick bi-layered walls which are c.2/3 of the total thickness. Micropyle is present, robust and protruding, and occasionally has a rounded polar body attached to the micropyle. Within the oocyst, a residuum, in addition, two to five polar granules are present. There are four ellipsoidal sporocysts 9-11 × 5-6 µm with flattened to half-moon shaped Stieda bodies. Sub-Stieda body and para-Stieda body are absent. The sporocysts contain sporocyst residuums composed of a few spherules scattered among the sporozoites. Within the sporozoites, anterior and posterior refractile bodies are present, but the nucleus is indiscernible. To further characterise the novel Eimeria species from H. longirostris, molecular analysis was conducted at the 18S ribosomal RNA locus, using PCR amplification and cloning. Two cloned sequences from the novel Eimeria were compared with those from other Eimeria spp. with the highest genetic similarity of 97.6% and 97.2% from Clone 1 and 2, respectively with Eimeria reichenowi (AB544308) from a hooded crane (Grus monacha Temminck) in Japan. Both sequences grouped in a clade with the Eimeria spp. isolated from wetland birds, which include Eimeria paludosa (KJ767187) from a dusky moorhen (Gallinula tenebrosa Gould) in Western Australia, Eimeria reichenowi (AB544308) and Eimeria gruis (AB544336) both from hooded cranes. Based on the morphological and molecular data, this Eimeria sp. is a new species of coccidian parasite and is named Eimeria haematopusi n. sp. after its host H. longirostris.

The putative vacuolar processing enzyme gene TaVPE3cB is a candidate gene for wheat stem pith-thickness.

KEY MESSAGE: The vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses. The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of 'Westonia' × 'Kauz'. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat 'Westonia'. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.

Next-generation sequencing amplicon analysis of the genetic diversity of Eimeria populations in livestock and wildlife samples from Australia.

Eimeria is an important coccidian enteric parasite that infects a wide range of hosts and can cause substantial economic losses in the poultry and livestock industries. It is common for multiple Eimeria species to infect individual hosts, and this can make species identification difficult due to morphological similarities between species and mixed chromatograms when using Sanger sequencing. Relatively few studies have applied next-generation amplicon sequencing (NGS) to determining the genetic diversity of Eimeria species in different hosts. The present study screened 408 faecal samples from a range of hosts including livestock and wildlife using a previously developed quantitative polymerase chain reaction (qPCR) at the 18S locus and conducted amplicon NGS on the positives using a ~ 455-bp fragment of the 18S locus. A total of 41 positives (10.1%) were identified by qPCR from various hosts and NGS was successful for 38 of these positives. Fifteen Eimeria species and three genotypes were detected by NGS: E. ferrisi, E. kanyana, E. potoroi, E. quokka, E. setonicis, E. trichosuri, E. reichenowi, E. angustus, E. ahsata, E. auburnensis, E. bovis, E. brasiliensis, E. christenseni, E. crandallis, E. ovinoidalis, Eimeria sp. (JF419345), Eimeria sp. (JF419349) and Eimeria sp. (JF419351). Mixed infections were detected in 55.3% (21/38) of positive samples. The most striking finding was the identification of the same species in different hosts. This could be due to contamination and/or mechanical transmission or may provide support for previous studies suggesting that Eimeria species can infect not just closely related hosts but different genera and further research is required. This is also the first study to audit Eimeria populations in livestock (sheep and cattle) by NGS and could be applied in the future to determine the extent of pathogenic species and outcomes of Eimeria control strategies.

Systemic Perturbations in Amine and Kynurenine Metabolism Associated with Acute SARS-CoV-2 Infection and Inflammatory Cytokine Responses.

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.

Incomplete Systemic Recovery and Metabolic Phenoreversion in Post-Acute-Phase Nonhospitalized COVID-19 Patients: Implications for Assessment of Post-Acute COVID-19 Syndrome.

We present a multivariate metabotyping approach to assess the functional recovery of nonhospitalized COVID-19 patients and the possible biochemical sequelae of "Post-Acute COVID-19 Syndrome", colloquially known as long-COVID. Blood samples were taken from patients ca. 3 months after acute COVID-19 infection with further assessment of symptoms at 6 months. Some 57% of the patients had one or more persistent symptoms including respiratory-related symptoms like cough, dyspnea, and rhinorrhea or other nonrespiratory symptoms including chronic fatigue, anosmia, myalgia, or joint pain. Plasma samples were quantitatively analyzed for lipoproteins, glycoproteins, amino acids, biogenic amines, and tryptophan pathway intermediates using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Metabolic data for the follow-up patients (n = 27) were compared with controls (n = 41) and hospitalized severe acute respiratory syndrome SARS-CoV-2 positive patients (n = 18, with multiple time-points). Univariate and multivariate statistics revealed variable patterns of functional recovery with many patients exhibiting residual COVID-19 biomarker signatures. Several parameters were persistently perturbed, e.g., elevated taurine (p = 3.6 × 10-3 versus controls) and reduced glutamine/glutamate ratio (p = 6.95 × 10-8 versus controls), indicative of possible liver and muscle damage and a high energy demand linked to more generalized tissue repair or immune function. Some parameters showed near-complete normalization, e.g., the plasma apolipoprotein B100/A1 ratio was similar to that of healthy controls but significantly lower (p = 4.2 × 10-3) than post-acute COVID-19 patients, reflecting partial reversion of the metabolic phenotype (phenoreversion) toward the healthy metabolic state. Plasma neopterin was normalized in all follow-up patients, indicative of a reduction in the adaptive immune activity that has been previously detected in active SARS-CoV-2 infection. Other systemic inflammatory biomarkers such as GlycA and the kynurenine/tryptophan ratio remained elevated in some, but not all, patients. Correlation analysis, principal component analysis (PCA), and orthogonal-partial least-squares discriminant analysis (O-PLS-DA) showed that the follow-up patients were, as a group, metabolically distinct from controls and partially comapped with the acute-phase patients. Significant systematic metabolic differences between asymptomatic and symptomatic follow-up patients were also observed for multiple metabolites. The overall metabolic variance of the symptomatic patients was significantly greater than that of nonsymptomatic patients for multiple parameters (χ2p = 0.014). Thus, asymptomatic follow-up patients including those with post-acute COVID-19 Syndrome displayed a spectrum of multiple persistent biochemical pathophysiology, suggesting that the metabolic phenotyping approach may be deployed for multisystem functional assessment of individual post-acute COVID-19 patients.

New insights into the evolution of wheat avenin-like proteins in wild emmer wheat (Triticum dicoccoides).

Fifteen full-length wheat grain avenin-like protein coding genes (TaALP) were identified on chromosome arms 7AS, 4AL, and 7DS of bread wheat with each containing five genes. Besides the a- and b-type ALPs, a c type was identified in the current paper. Both a and b types have two subunits, named x and y types. The five genes on each of the three chromosome arms consisted of two x-type genes, two y-type genes, and one c-type gene. The a-type genes were typically of 520 bp in length, whereas the b types were of 850 bp in length, and the c type was of 470 bp in length. The ALP gene transcript levels were significantly up-regulated in Blumeria graminis f. sp. tritici (Bgt)-infected wheat grain caryopsis at early grain filling. Wild emmer wheat [(WEW), Triticum dicoccoides] populations were focused on in our paper to identify allelic variations of ALP genes and to study the influence of natural selection on certain alleles. Consequently, 25 alleles were identified for TdALP-bx-7AS, 13 alleles were identified for TdALP-ax-7AS, 7 alleles were identified for TdALP-ay-7AS, and 4 alleles were identified for TdALP-ax-4AL Correlation studies on TdALP gene diversity and ecological stresses suggested that environmental factors contribute to the ALP polymorphism formation in WEW. Many allelic variants of ALPs in the endosperm of WEW are not present in bread wheat and therefore could be utilized in breeding bread wheat varieties for better quality and elite plant defense characteristics.

Morphological and genetic characterization of the first Isospora species (I. lugensae n. sp.) from a Kerguelen petrel (Lugensa brevirostris).

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.

Yield-Related QTL Clusters and the Potential Candidate Genes in Two Wheat DH Populations.

In the present study, four large-scale field trials using two doubled haploid wheat populations were conducted in different environments for two years. Grain protein content (GPC) and 21 other yield-related traits were investigated. A total of 227 QTL were mapped on 18 chromosomes, which formed 35 QTL clusters. The potential candidate genes underlying the QTL clusters were suggested. Furthermore, adding to the significant correlations between yield and its related traits, correlation variations were clearly shown within the QTL clusters. The QTL clusters with consistently positive correlations were suggested to be directly utilized in wheat breeding, including 1B.2, 2A.2, 2B (4.9-16.5 Mb), 2B.3, 3B (68.9-214.5 Mb), 4A.2, 4B.2, 4D, 5A.1, 5A.2, 5B.1, and 5D. The QTL clusters with negative alignments between traits may also have potential value for yield or GPC improvement in specific environments, including 1A.1, 2B.1, 1B.3, 5A.3, 5B.2 (612.1-613.6 Mb), 7A.1, 7A.2, 7B.1, and 7B.2. One GPC QTL (5B.2: 671.3-672.9 Mb) contributed by cultivar Spitfire was positively associated with nitrogen use efficiency or grain protein yield and is highly recommended for breeding use. Another GPC QTL without negatively pleiotropic effects on 2A (50.0-56.3 Mb), 2D, 4D, and 6B is suggested for quality wheat breeding.

Morphological and genetic characterization of Eimeria chalcoptereae n. sp. (Apicomplexa: Eimeriidae) in a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia.

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).

Molecular and morphological analysis of a Caryospora-like isolate (Apicomplexa: Eimeriidae) from the magpie-lark (Grallina cyanoleuca) (Latham, 1801) in Western Australia.

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.

Wheat grain protein accumulation and polymerization mechanisms driven by nitrogen fertilization.

In wheat (Triticum aestivum) grain yield and grain protein content are negatively correlated, making the simultaneous increase of the two traits challenging. Apart from genetic approaches, modification of nitrogen fertilization offers a feasible option to achieve this aim. In this study, a range of traits related to nitrogen-use efficiency in six Australian bread wheat varieties were investigated under different nitrogen treatments using 3-year multisite field trials. Changes in the individual storage protein composition were detected by high-performance liquid chromatography. Our results indicated that wheat grain yield and grain protein content reacted similarly to nitrogen availability, with grain yield being slightly more sensitive than grain protein content, and that genotype is a vital determinant of grain protein yield. Measurement of the glutamine synthetase activity of flag leaves and developing grains revealed that high nitrogen availability prompted the participation of glutamine in biological processes. In addition, a more significant accumulation of gluten macropolymer was observed under the high-nitrogen treatment from 21 days post-anthesis, and the underlying mechanism was elucidated by a comparative proteomics study. A yeast two-hybrid experiment confirmed this mechanism. The results of this study revealed that peptidyl-prolyl cis-trans isomerase (PPIase) was SUMOylated with the assistance of small ubiquitin-related modifier 1 and that high nitrogen availability facilitated this connection for the subsequent protein polymerization. Additionally, luminal-binding protein 2 in the endoplasmic reticulum played a similar role to PPIase in the aggregation of protein under high-nitrogen conditions.

Isospora coronoideae n. sp. (Apicomplexa: Eimeriidae) from the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758) in Western Australia.

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).

Further characterisation of Leucocytozoon podargii in wild tawny frogmouths (Podargus strigoides) in Western Australia.

The present study assessed the prevalence and morphology of Leucocytozoon podargii from wild tawny frogmouths (Podargus strigoides) in Western Australia (WA) and genetically characterised the cytochrome b gene (cyt b) of L. podargii in wild tawny frogmouths from WA and Queensland (QLD). The prevalence of L. podargii in wild tawny frogmouths from WA was 93.3% (14/15; 95% CI, 68.1-99.8%). The morphological characters of L. podargii from WA were similar to L. podargii from QLD: the gametocytes were round-oval shape, approximately 8-12 µm in diameter; the macrogametocytes were 12.4 µm in diameter; microgametocytes were 10.4 µm in diameter; and the ratio of macrogametocytes and microgametocytes was 3:2. Sequence analysis of partial cyt b gene fragments revealed that L. podargii sequences isolated from wild tawny frogmouths in WA shared the highest similarity (99.8% at nucleotide level and 100% at protein level) with L. podargii isolated from wild tawny frogmouths in QLD. The mitochondrial 18S rRNA gene of L. podargii gametocytes was quantified using droplet digital PCR (ddPCR), and the highest gametocyte load was detected in the lung. This finding corresponds to the results of the histological study. Based on the morphological and molecular studies, it was concluded that the Leucocytozoon parasite identified from wild tawny frogmouths in WA is consistent with L. podargii from wild tawny frogmouths in QLD, and the present study has genetically characterised two different L. podargii genotypes (QLD and WA) for the first time.

Identification of a novel species of Eimeria Schneider, 1875 from the woylie, Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials.

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. oöcysts 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.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

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 ß-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.

Morphological and molecular characterisation of Isospora butcherae n. sp. in a silvereye (Zosterops lateralis) (Latham, 1801).

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.

Gastrointestinal helminths in farmers and their ruminant livestock from the Coastal Savannah zone of Ghana.

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.

Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Chronic Cystoisospora belli infection in an immunocompetent Myanmar refugee - microscopy is not sensitive enough.

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.

Morphological and molecular characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa: Eimeriidae, 1989, Paperna and Landsberg) in a western bearded dragon (Pogona minor minor).

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.