Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids. Candida metapsilosis, an emerging fungal pathogen of growing concern. We found that all isolates were the result of a single hybridization between two unidentified species, which points to hybridization as a mechanism for the origin of a virulent lineage. We found that the hybrids likely originated by sexual reproduction as they were diploids and retained genomic regions of opposite mating types. We reconstructed the aftermath of the genome merging by identifying where recombination led to the removal of one of the parental subgenomes. We finally compare the newly-sequenced genome with those of other pathogens from the Candida clade and establish global trends, such an enriched repertoire in cell-wall proteins in more virulent species. Our results provide insight into how hybridization may play a key role in the emergence of novel pathogenic lineages.Human pathogens belong to different phylogenetic clades and it is clear that the ability to infect humans emerged several times independently. The sequencing and comparison of genomes from pathogenic and non-pathogenic species and strains paves the way to identify what genomic changes underlie the emergence of virulence. In this study we sequenced 11 globally-distributed clinical isolates of  Candida parapsilosis species complex.Hybridization between species is an important evolutionary mechanism that can drive the origin of novel lineages and adaptation to new environments. Hybridization results in the combination of two diverged genomes, which are subsequently shaped by processes of recombination, deletion, and other genomic rearrangements. Genomics have recently paved the way to investigate the stochastic and adaptive processes that follow genomic hybridization. As compared to metazoans or plants, fungi have lower prezygotic barriers and can reproduce clonally for long periods of time, thus hybridization is thought to have a large impact in the evolution of this clade. Consistently, the presence of hybrids in fungi have been extensively documented and an increasing number of cases are being described in the literature –8. HybriC. parapsilosis groups I, II and III, have been re-defined as different species: C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, respectively [C. metapsilosis being the one with the lowest clinical prevalence, and accounting for only 1.1 to 8.4% of the infections caused by the complex [C. parapsilosis sensu lato species, and particularly little is known about the rarer species C. orthopsilosis and C. metapsilosis. Nonetheless, results of several in vitro and in vivo studies suggest that, consistent with its lower prevalence, C. metapsilosis is the least virulent species of the complex [C. parapsilosis sensu lato isolates, revealed that C. metapsilosis strains were unable to produce extracellular lipases and form pseudohyphae, attributes that are both recognized as important virulence factors for Candida spp. [C. metapsilosis isolates are killed more efficiently by and are less cytotoxic to human primary macrophages [This species complex comprises opportunistic pathogen species that cause serious infections in immunocompromised patients, and whose incidence has significantly increased in recent years . Three dectively . These s complex . In addi complex –15. Ther complex –20. A reida spp. . Furtherrophages ,18. Finarophages ,21,22.C. parapsilosis sensu lato spp. underlines the importance of studies investigating the virulence attributes and molecular genetics of these species. Furthermore, the C. parapsilosis species complex offers an exquisite system for the study of the evolution of pathogenic yeasts and their adaptation to the human host. The complex belongs to the broader CTG clade of Saccharomycetales, which include species that decode the CUG codon as serine instead of leucine. Although the CTG clade includes other clinically important species such as C. albicans and C. tropicalis [Candida parapsilosis species complex evolved pathogenesis towards humans independently of C. albicans and their closest relatives. The sequencing of reference strains for C. parapsilosis [C. orthopsilosis [C. albicans. Such analyses have revealed that, whereas most Candida species display a similar content of families represented by singleton genes, most of the variability is related to copy number differences in multi-gene families, with pathogens having increased number of members in families related to virulence [C. parapsilosis has an expanded Hyr/Iff family of virulence-related cell wall genes relative to the less virulent species C. orthopsilosis . Subsequent analyses have assessed the genomic diversity among C. parapsilosis [C. orthopsilosis [C. parapsilosis, pointing to several recent and recurrent clinical outbreaks from the environment [C. orthopsilosis subspecies, including the description of one virulent hybrid lineage isolated from two very distant locations [C. parapsilosis sensu lato species, for C. metapsilosis we lack both a reference genome and a comprehensive insight on the genomic diversity across isolates. This situation precludes understanding the emergence of virulence traits within this complex. To fill in this important gap we undertook the sequencing and analysis of the genomes of eleven C. metapsilosis clinical isolates. Unexpectedly, we found that all sequenced isolates, sampled from geographically distant locations, presented highly heterozygous genomes, which we show to result from a single hybridization event between two parental lineages differing by 4.5% at the nucleotide level.The growing incidence of infections due to opicalis , their ppsilosis  and C. opsilosis  has beenirulence . For inspsilosis  and C. opsilosis  isolatesironment , or the ocations . In contC. metapsilosis clinical isolates from different geographical locations , corresponding to the template domain of the telomerase (TER1) [C. metapsilosis, which is roughly similar to the gene counts in C. parapsilosis  and C. orthopsilosis .The final assembly resulted in a total size of 13.3 Mb in seven putative chromosomes and two unplaced scaffolds, which is similar to the number of bands observed in a Pulsed Field Gel Electrophoresis (PFGE) . The obse (TER1)  in a regCandida parapsilosis complex generally display linear mitochondrial chromosomes, but rare isolates presenting circularized mitochondrial DNA (mtDNA) have been identified in C. orthopsilosis and C. metapsilosis [C. metapsilosis strain bearing a circular mitochondrial chromosome [in-silico predictions were confirmed experimentally through PCR and PFGE tests  [C. metapsilosis hybridization is a more recent event or, alternatively, that C. orthopsilosis lost heterozygosity more rapidly.We will refer to blocks of homozygosity as loss of heterozygosity blocks . Such LOH blocks could be the result from several basic mechanisms, including mitotic recombination, break-induced replication or gene conversion . Alternaas used) . This diC. orthopsilosis  of LOH blocks, with 1,581 LOH blocks having identical boundaries across all sequenced strains  as compared to 2,151 bp in psilosis . This diC. orthopsilosis case, where a completely homozygous strain  exists [C. metapsilosis strains is homozygous. Furthermore, both haplotypes in a given heterozygous region are roughly equally distant to either C. orthopsilosis or C. parapsilosis out-groups . This prevents assignment of each haplotype in a heterozygous region to a particular parent, and prevents phasing the homozygous regions  is present in homozygous regions. We applied this procedure to scaffold6 from PL448, which underwent LOH of the entire chromosome (Contrary to the ) exists , none of regions . In the romosome . This prromosome .C. orthopsilosis hybrid with similar divergence between parents [Pichia sorbitophila has led to more unbalanced inheritance in that species [Finally, to study the pattern of LOH on a local scale, we examined one specific region in four strains by PCR and re-sequencing. This 3.6 kb-long region around the gene g3863.t1 (coding for a protein with a DEAD-box RNA helicase domain), is interesting because it encompasses eight recent LOH blocks that are present specifically in 1 to 3 of the four strains. We can consider these eight LOH blocks to have originated independently and we can assess whether the same or a distinct parental haplotype was introgressed in each of these by selectively amplifying and sequencing each of the DNA molecules. Notably, in all cases LOHs present in a given strain resulted from haplotype A overwriting haplotype B  Figs. Th species .C. metapsilosis strains  suggested a diploid state for the sequenced  strains . In addi strains . These a strains . FACS  has overwritten a nonhomologous 6 kb region that is normally present in MTLa and contains PAPa, OBPa and PIKa (in that order). Consistent with this introgression happening after the single hybridization event, the divergence between alleles in the introgressed region is very low (0.025%). These results were confirmed by PCR amplification and re-sequencing of five representative strains , DNS100 (CP376)). The resulting haplotype contains an intact copy of MTLalpha2 as well as MTLa2 and MTLa1, it has no PAP gene of any kind, and its PIK gene is chimeric  in sequenced strains were subjected to restrictive manual curation see , resultiC. metapsilosis clinical isolates we employed several alternative strategies. First, multiple correspondence analyses (MCA) was conducted using the SNPs  suggests that a limited number of point mutations accumulated during this time: 3 SNPs in the mitochondrial genome (0.0124% divergence) and 53 SNPs in the longest common homozygous region (0.01767%).The significant differences between the hybrid isolates and the certain degree of geographical structure suggest that this lineage is relatively ancient and spread globally a long time ago. This is in stark contrast with what has been found for the dentical . Given oC. metapsilosis allows, for the first time, to perform a comprehensive comparative genome analysis of the entire C. parapsilosis species complex. Whole genome alignments show that synteny is largely conserved among the three species of the complex  than either of them to C. metapsilosis . We compared the gene content of the three species and compared them with other 23 sequenced Saccharomycetes by reconstructing the complete collection of evolutionary histories of the genes encoded in their genomes (i.e. the phylome) and establishing orthology and paralogy relationships among them [Candida species , of which 5,045 (88%) are present across all three species. Of the widespread groups, 4,574 (91%) were present as one-to-one orthologs, while 226, 107 and 124 groups contained C. metapsilosis, C. orthopsilosis and C. parapsilosis specific paralogs, respectively. Differences in gene content for the three species are presented in , than C. parapsilosis (17), but more than C. orthopsilosis (3 in 90–125 and 4 in MCO456) (see Phy00767CU tree in PhylomeDB). Interestingly, C. metapsilosis encodes one ALS gene, which is very close to ALS6 (CPAG_05054/CPAR2_404790), while the two C. orthopsilosis strains encode an ortholog closer to ALS7 . Thus the lack of some ALS genes but not the number of members of Hyr/Iff family correlates with the inability to produce pseudohyphae and consequent lower virulence than in the remaining C. parapsilosis complex species.Predicted genes in the three species from the ented in . We hereented in . The abidohyphae , and thiC. albicans and C. parapsilosis [C. metapsilosis strains were unable to produce extracellular lipases [C. parapsilosis and C. orthopsilosis (4). This indicates that the observed phenotypic differences may be due to different regulation of the lipase activity rather than to an inherent inability to produce secreted lipases. It has been demonstrated that individual lipase genes are differentially regulated in C. albicans during infection [C. metapsilosis lipase genes, it is appealing to speculate that a similar phenomenon exists in this species. Secreted aspartic proteases are also considered an important virulence factor in C. albicans and C. parapsilosis [Candida parapsilosis complex have also been shown to exhibit this activity [C. metapsilosis and C. parapsilosis encode more (14) secreted aspartic proteases (SAP) than any other Candida spp.: C. orthopsilosis (11 in MCO456 and 11 in 90–125) or C. albicans (10) , that are important for iron acquisition, than C. albicans (5). Overall, the broad functional class corresponding to cell wall and adhesion proteins seems to have a tendency to show larger numbers in pathogenic species as compared to less pathogenic ones, and is the only broad functional class that seems also expanded in non-CTG yeast pathogens, such as those in the Nakaseomyces clade [We and others have previously shown that secreted lipases play an important role in the virulence of psilosis ,40–42. D lipases , we founnfection ,44, and psilosis ,46, and activity . Intereses clade .C. metapsilosis is a diploid hybrid species resulting from a single hybridization event, likely through mating, of two parental lineages that were ~4.5% divergent. This hybrid lineage expanded globally and currently isolated strains differ to a significant extent in their genomic background. Divergence has been mostly driven by differential LOH events but also by lineage-specific copy number variations, including large partial aneuploidies. Earlier studies based on AFLP have previously described a high degree of genetic heterogeneity among C. metapsilosis strains [C. metapsilosis (22–26 SNPs/kb), are much larger than those described in C. albicans (2.5–3 SNPs/kb) [C. albicans- and perhaps other highly heterozygous species in the CTG clade—may have originated via a similar process a longer time ago.Our results show compelling evidence that the opportunistic pathogen  strains , which iSNPs/kb) . HoweverC. parapsilosis clade. We propose the existence of at least five different homozygous lineages and at least two hybrid lineages resulting from distinct combinations of the former. Two of the five homozygous lineages would be nowadays represented by C. parapsilosis and homozygous C. orthopsilosis Type 2 strains such as 90–125, whereas the remaining three are only partially represented by the sequences of C. orthopsilosis MCO456 and the C. metapsilosis sequences presented in this work  in 2 ml YPD medium (0.5% (w/v) yeast extract, 1% (w/v) peptone, 1% (w/v) glucose) supplemented with 100 unit/ml penicillin-streptomycin solution (Sigma). Subsequently, cells were centrifuged  and were washed twice with 1x sterile PBS. The pellet was resuspended in 500 μl lysis buffer (1% (w/v) SDS, 50 mM EDTA, 100 mM TRIS pH = 8), 500 μl glass bead was added to the cells and were disrupted by using a vortex for 3 minutes. 275 μl 7M ammonium-acetate was added  and the samples were then cooled on ice for 5 minutes. 500 μl of chloroform-isoamylalcohol (24:1) was added to the mixture, and the samples were centrifuged for 10 minutes at 16000 xg. The upper phase was transferred to a new microcentrifuge tube, and the previous step was repeated. 500 μl isopropanol was mixed with the upper phase in a new microcentrifuge tube, and the mixture was held in a refrigerator at -20°C for 5 minutes. The solution was centrifuged at 16000 xg for 10 minutes. The supernatant was discarded, and the pellet was washed twice with 500 μl 70% ethanol. After the second washing step the pellet was dried, and resuspended in 100 μl sterile bi-distilled water containing 250 μg/ml RN-ase (Sigma).The genome sequences for the 11 strains were obtained at the Ultra-sequencing core facility of the CRG, using Illumina GAIIx, HiSeq2000 and MiSeq sequencing machines. For paired-end libraries, DNA was fragmented by nebulization or in Covaris to a size ~300 bp, ~400 bp, ~600 bp. The ends of the DNA fragments were blunted with T4 DNA polymerase and Klenow fragment (New England Biolabs), after shearing. DNA was purified with a QIAquick PCR purification kit (Qiagen). 3’-adenylation was performed by incubation with dATP and 3’-5’-exo- Klenow fragment (New England Biolabs). DNA was purified using MinElute spin columns (Qiagen) and double-stranded Illumina paired-end adapters were ligated to the DNA using rapid T4 DNA ligase (New England Biolabs). After another purification step, adapter-ligated fragments were enriched, and adapters were extended by selective amplification in an 18-cycle PCR reaction using Phusion DNA polymerase (Finnzymes). Libraries were quantified and loaded into Illumina flow-cells at concentrations of 7–20 pM. Cluster generation was performed in an Illumina cluster station. Sequence runs of 2x50, 2×76, 2x100 or 2x250 cycles were performed on the sequencing instrument. For the preparation of mate-pair libraries 15 micrograms of genomic DNA were sheared using a covaris instrument to the desired size range of 2.5 or 5 kb, respectively. Following size selection on a 0.8% agarose gel, the size fraction of interest was recovered, and library preparation was performed using a modification of the Illumina mate-pair preparation protocol, whereby a biotinylated double-stranded adapter was included in the circularisation reaction . After cC. parapsilosis complex species (~13 Mb in 7–8 chromosomes), we assumed the presence of heterozygous regions in our strains. Heterozygous regions (9 Mb in 861 contigs) were removed by Haplomerger version 20120810 [Reads were pre-processed before assembly to trim at the first undetermined base or at the first base having PHRED quality below 10. We filtered out pairs with one (or both) reads shorter than 31 bases after trimming. SOAPdenovo2  was used20120810 . Subsequ20120810  and gapsC. parapsilosis CDC317 gene models for training [Candida albicans or Saccharomyces cerevisiae, based on predictions from the MetaPhORs approach [Genes were predicted using Augustus version 2.5.5  and C. ptraining . Predictapproach . Finallyapproach .C. metapsilosis assembly using Bowtie2 with “very sensitive local alignment” mode [100) and the expected number of LOH blocks of this length or longer in a 13.6 Mb genome is approximately 5,854 blocks, these numbers vary dramatically to 0.0001 and 58 when a 200 bp threshold is applied . We compared the number of expected blocks of a given size or longer with the observed sizes in the real strains  with the homozygous chromosome set of C. metapsilosis assuming 4.5% divergence between haplotypes. LOH regions (at least 100bp regions having less than 2 SNPs) appear by chance in all simulations and on average sum up 782,850 bp (5.84% of the genome). Thus we estimate that less than 6% of the regions detected as LOH in our analysis correspond to false positives. Increasing LOH length cut-off from 100bp to 200bp decreases false positive rate to 0.13%, however. Throughout the manuscript we provide estimates based on both of these thresholds. The sequence divergence between the parental haplotypes was calculated as the number of heterozygous sites found in heterozygous regions divided by the cumulative length of heterozygous regions, using a 100 bp threshold.Genomic reads were aligned onto nt” mode . SNPs annt” mode . We filt strains . This main silico differentiation of linear and circular mtDNA chromosomes in C. metapsilosis. First, due to the presence of telomeres, consisting of tandem arrays of 620 bp repeat, linear chromosomes are expected to be longer  than circular ones  [C. metapsilosis MCO448 (NC_006971) as indicated above.Two main characteristics allow Y391853) . Second,C. metapsilosis isolates were analysed by PFGE and PCR essentially as described in [cox2 and nad4. For PCR analysis, the reactions contained diluted total cell DNA, 0.5 μM upstream and downstream primers , 0.2 mM dNTPs each, 1× reaction buffer with 2 mM MgCl2, and 1 U of Taq DNA polymerase (Invitrogen). The amplification was performed using the following cycler profile: 3 min at 95°C; 25× ; 3 min at 72°C. The primers bind to the opposite subterminal regions of the linear mitochondrial genome and allow amplification of about 0.95 kb long fragment derived from the end-to-end junction of circularized genome forms . bam2sv.py v1.0 detects duplications, deletion and inversions by means of insert size deviations and incongruent read pairing between paired-end reads. In addition, duplications and deletions are detected from deviations from mean depth of coverage. All detected variants were manually curated. In addition, we generated genome graphs for all chromosomes illustrating copy number variation, as well as, heterozygous and homozygous regions , CP61 (DNS94), CP376 (DNS100) and PL429. Four PCR primers were designed using Primer3 v4 webtool [To validate the two parental sequences, we performed PCR and Sanger sequencing of the region scaffold2|size2959145:1,570,477–1,574,155 from  webtool , namely FWD_1: 5’-TTGACTGCTGAAGCTGTCTTGG-3’;FWD_2: 5’-TTAACCGCTGAAGTTGTCTTTGA-3’;REV_1: 5’-ATTCCATCTTGGCGCATCTT-3’;REV_2: 5’-GACTCCATCTTGACGAATCTTGG-3’.Thus, four touchdown PCR reactions  were carried out using Expand Long Range, dNTPack kit (Roche) according to manufacturer’s instructions. Briefly, each reaction included primer concentration of 0.3 μM, 10 μl of 5X Buffer with MgCl2 , 2.5 μl of PCR Nucleotide Mix, 3% (v/v) of DMSO, 100 ng of DNA and 3.5 U of Enzyme Mix in a final volume of 50 μl. Cycling condition began with a warm-up step of 2 min at 92°C, followed by 15 cycles of 10 seconds at 92°C, 15 seconds at the corresponding annealing temperature per each pair of primers (decreasing 0.5°C each cycle) and 4 min at 68°C. The initial annealing temperatures were: 61.6°C, 64.1°C, 60°C, and 62.5°C for FWD_1+REV_1, FWD_1+REV_2, FWD_2+REV_1 and FWD_2+REV_2, respectively. Then, other 20 cycles of 10 seconds at 92°C, 15 seconds at the corresponding annealing temperature and 4 min at 68°C (increasing the extension time 20 seconds at each cycle) were set up, with a final extension step at 68°C for 7 minutes. PCR products were confirmed by 1.5% agarose gel electrophoresis and were then purified using QIAquick PCR Purification Kit (QIAGEN). Specific PCR products were only obtained when combining the primer sets FWD_1+REV_2, and FWD_2+REV_1 (amplicon size of 3678 bp), while no product was obtained when combining FWD_1+REV_1 or FWD_2+REV_2 primers . The purINT_FWD: 5’- AGTTTGGAAATTGTCATCTTGGAT-3’;INT_REV: 5’- ACTTCTTTAAAATACTAACCACATCCATCTC-3’.C. metapsilosis: SZMC21154, SZMC8092, SZMC8094, SZMC8095, CP61 (DNS94), CP367 (DNS100). We have designed 8 different primers:The MTL introgression was checked by means of PCR and Sanger sequencing in following strains of MTLα1_1F: 5’-GCCGCCTGAGAAGTATGAAG-3’;MTLα1_1R: 5’-TTGGTGACCACAGGAAAACA-3’;MTLα2_1F: 5’-GTGCTCCTCAAGCACAATCA-3’;MTLα2_1R: 5’-GTCGCCCAGACAACTCTAGC-3’;MTLa1_1F: 5’-GCTTGAGTGGGGATTGAGTC-3’;MTLa1_1R: 5’-AATCGTTTTCGGGGTTTTCT-3’;MTLa2_1F: 5’-TCTCCGGATTCGTTCAATTC-3’;MTLa2_1R: 5’-CTTGACCCCAAAGCTTTCAA-3’.The introgression was validated by 4 different PCRs  using Pfu DNA polymerase from PROMEGA. The reaction mixture consisted of 5μl of Buffer 10X with MgSO4 and1μl of 10 mM dNTPs, both provided by the manufacturer, 2μl 10 μM of both forward and reverse primers, 0.4μl of Pfu DNA polymerase 3 U/μl and water filled up to 50μl. The PCR started with initial denaturation at 95°C for 2min, this was followed 30 cycles of 30 seconds at 92°C, 30 seconds at 61.0°C, 30 seconds at 72°C, it was finished with final extension for 5min at 72°C and cooled to 4oC. PCR products were confirmed by 1.5% agarose gel electrophoresis and were then purified using QIAquick PCR Purification Kit (QIAGEN).Finally, in order to confirm linearity of MTL after introgression, we have amplified and Sanger sequenced genomic regions flanking introgression site. We have designed 6 primers:P1f: 5’-TATCGGGTTTGAAGCTGCTC -3’;P1rα: 5’-GCTGAATGCTGGTTTTTGGT -3’;P2ra: 5’-TACTCCACGTTGTTTTGTTAAAG-3’;P2fα: 5’-TTATGATTGGGATGGGTTGG -3’;P2fa: 5’-CACATTTGAATGGACGTTGG -3’;P2r: 5’-CGATAAATCAGCGCAACAAT-3’.The linearity of introgression was validated by 4 different PCRs  using Pfu DNA Polymerase from PROMEGA. The reaction mixture consisted of 5μl of Buffer 10x with MgSO4 and1μl of 10 mM dNTPs, both provided by the manufacturer, 2μl 10 μM of both forward and reverse primers, 0.4μl of Pfu DNA polymerase 3 U/μl and water filled up to 50μl. The PCR started with initial denaturation at 95°C for 2min, this was followed 30 cycles of 30 seconds at 92°C, 30 seconds at either 56.2°C (P1f ‐ P1rα), 57.2°C (P1f –P2ra) or 54.2°C (P2fα–P2r and P2fa–P2r); 30 seconds at 72°C, it was finished with final extension for 5 min at 72°C and cooled to 4oC. PCR products were confirmed by 1.5% agarose gel electrophoresis and were then purified using QIAquick PCR Purification Kit (QIAGEN). Sanger sequencing was performed using an ABI Prism 3730xl DNA Analyzer (Applied Biosystems).8 cells were resuspended in Isotonic Buffer  containing 1 M potassium-thioglycolate , and it was incubated for 1 hour, at 30°C in an orbital shaker (180 rpm). The suspension was washed once with Isotonic Buffer . The pellet was resuspended in 5 ml Isotonic Buffer containing 3% (w/v) Helicase and 0.5% (w/V) NovoZym 234 (Novo BioLabs), and it was incubated at 30°C, in a sterile 15 ml Falcon tube, overnight, in an orbital shaker (180 rpm). The spheroplasts were collected and washed once with Isotonic Buffer . The pellet was resuspended in 42°C 0.125 M EDTA, mixed immediately with prewarmed (42°C) 2% (w/v) low-melting-point agarose (Sigma), then placed into a mould chamber. After solidification, inserts were incubated in 2 ml NDS buffer (1% (w/v) N-laurylsarcosine in 0.5 M EDTA, pH = 9.5) supplemented with 1 mg/ml Proteinase K (Sigma) at 50°C. During the two days of incubation the NDS-Proteinase K solution was replaced once. The inserts were washed once with 0.5 M EDTA (pH = 8) overnight, then were stored in 0.5 M EDTA (pH = 8) at 4°C until usage. Finally, yeast chromosomes were separated by CHEF [Yeasts cells were cultured for 24 hours, at 30°C, in an oxygen rich environment, in 5ml YPD (0.5% (w/v) yeast extract, 1% (w/v) peptone, 1% (w/v) dextrose) supplemented with 100 unit/ml penicillin-streptomycin (Sigma) in an orbital shaker (180 rpm). After 24 hours 100 μl suspension was transferred to 5 ml YPD and the cells were incubated for 24 hours again, under the same conditions. Agarose blocks containing the intact chromosomes were prepared using the method of Schwartz & Cantor  with the by CHEF  method bCandida samples. Cells were grown in YPD medium at 30°C , harvested, resuspended in deionized distilled water and fixed in ethanol at a 107 cells/ml concentration . For the staining of cells with SYBR Green I, cells were first washed and resuspended in 750 μl of 50 mM sodium citrate buffer, treated with 250 μl of 1mg/ml RNase A solution for 1 hour at 50°C and finally with 50 μl of 20 mg/ml proteinase K solution for 1 hour at 50°C. Then, 20 μl of SYBR Green I  were added to the samples and they were stained overnight at 4°C protected from light. Triton X-100 was added at a final concentration of 0.25% (v/v) and samples were vortex. Finally, samples were sonicated  to eliminate most of the cell clumps before FACS analysis with FACScan at the FACS Unit from CRG/UPF.Ploidy analyses were confirmed using FACS for 12 C. parapsilosis CDC317, C. orthopsilosis 90–125 and C. metapsilosis SZMC8094 were aligned with LAST aligner v189 [Chromosomes/scaffolds of ner v189 . AlignmeC. metapsilosis protein-coding genes were reconstructed in the context of 27 Saccharomycotina species  Patterns from homozygous SNPs present in the longest LOH block that is shared by all the strains; (ii) SNP patterns from the whole genome. Heterozygous SNPs were encoded using the base which is alternative to that in the reference, and choosing randomly one of the alternative bases in heterozygous SNPs if both were different from the reference; and (iii) Haplotype patterns based on three-state haplotype  assignment in windows of 1 kb, resulting in 1,798 phylogenetically informative patterns; Separately, character-based matrices were reconstructed using (iv) 127 ploidy patterns from 84 deletions and 87 duplications ; ploidy e from R .Candida species and relatives. First a parsimony-based super-tree was reconstructed using duptree v1.48 [C. metapsilosis phylome available at PhylomeDB id 243 [Two different approaches were used to reconstruct a species tree of ee v1.48  from theB id 243 . Then a B id 243  and the B id 243 .http://phylomedb.org) as PhylomeDB ID 243.Sequencing data, genome assembly and annotation have been deposited to EBI-ENA (accession: PRJNA238968). Reconstructed trees and alignments were deposited to PhylomeDB . Two C. parapsilosis strains were included as reference. Chromosomes with potential rearrangements are denoted with yellow asterisks. Sequenced strains are marked with black asterisks.The chromosomes of several (PDF)Click here for additional data file.S2 FigCandida metasilosis MCO448 (NC_006971). Telomeres (marked with blue squares) are expected only in linear chromosomes, thus strains with circular mitochondrial chromosome (PL448 and SZMC21154) miss parts of telomeres. In addition, read pairs aligned on the ends of the reference present discordant pairing (marked in green) in strains with circular mitochondrial chromosome. B. The most of C. metapsilosis strains examined in this study contain a linear mitochondrial genome with the length of about 23 kbp . The genes atp6 and nad3 are located in the right and left subterminal region, respectively, and the telomeres on both ends of linear DNA molecules consist of a subterminal repeat  and a tandem repeat array . The STR contains a sequence cluster rich in guanine and cytosine residues , which is presumably involved in the mitochondrial telomere maintenance via homologous recombination . In contrast, the strains PL448 and SZMC21154 contain circular mitochondrial genomes that lack the right telomere and most of the tandem repeat array of the left telomere. The sequence analysis indicates that these genomes represent circularized mutants resulting form end-to-end fusions of originally linear DNA molecules. Since the sites where the fusion events occurred are different, we assume that these mutants emerged independently.A. Genomic reads from all strains were aligned on the linear mitochondrial chromosome reference of (PDF)Click here for additional data file.S3 FigC. metapsilosis PL429 (blue) and C. orthopsilosis MCO 456 (grey). Only LOH shorter than 10 kb are shown. Note, Y axis is log-scaled.Histogram of LOH block sizes for (PDF)Click here for additional data file.S4 FigScatter plot representing the size of loss of heterozygosity (LOH) regions and their position on the chromosome given as percentage of chromosome length. Colors represent dots density. Note, two replicas for PL429 (pe300 and pe600) are given in two separate panels.(PDF)Click here for additional data file.S5 FigDetails for the independent amplification of an heterozygous region (3678 bp) from two homologous chromosomes. A. Sets of primers  targeting known heterozygous sequences that flank the region of interest were designated. Forward  and reverse  primers differ by three SNP, so all primers align uniquely to a specific homologous chromosome. Four PCR reactions were performed for each strain. For these only two PCR reactions gave a product (positive controls): P1 obtained from combination of F1 and R2; and P2 from F2 and R1. The other two combinations of primers (F1R1 and F2R2) did not work as the primers anneal on homologous chromosome and not on the same chromosome (negative controls). B. Obtained PCR products are too larger to be sequence by single Sanger reaction. We thus have designed two additional primers  that target homozygous region in the middle of PCR products, so they can be used with both PCR products. For each PCR product  we performed four Sanger sequencing reactions:∘pos4_F1 primer amplification (F1),∘pos4_R2 primer amplification (R2),∘pos4_M1F (M1F_12),∘pos4_M1R (M1R_12)For F1R2 amplification product (P1):∘pos4_F2 primer amplification (F2),∘pos4_R1 primer amplification (R1),∘pos4_M1F (M1F_21),∘pos4_M1R (M1R_21)For F2R1 amplification product:(PDF)Click here for additional data file.S6 FigTwo forward primers and two reverse primers, with four and three different bases among them, respectively, corresponding to allelic differences in each parental sequence/genotype were designed. Thus, four touchdown PCR reactions  were carried out. Specific PCR products were only obtained when combining the primer sets FWD_1+REV_2, and FWD_2+REV_1 (amplicon size of 3678 bp), while no band was seen when combining FWD_1+REV_1 or FWD_2+REV_2 primers.(PDF)Click here for additional data file.S7 FigC. metapsilosis strains: BP57 (DNS25), CP61 (DNS94), CP367 (DNS100) and PL429. A) Following features are given for each strain: genomic read density and alignments, followed by aligned Sanger sequences from PCR product 1 (P1) and product 2 (P2). LOH events are marked by red rectangles or red arrow. The recombination donor is marked by green arrow. All recombinations in the analysed region originated from PCR product 2 (P2). B) Schematic representation of LOH events. Six LOH events (#1-#6) are common to three strains , while the remaining two (#7 and #8) are present only in PL429.In order to test whether LOH donor is randomly or preferentially selected, we have analysed in detail 3.6 kb genomic region  harboring eight LOH in four (PDF)Click here for additional data file.S8 FigC. metapsilosis strains. (A) We observed single peak close to 50% for all chromosomes of C. metapsilosis PL429, indicating it is diploid. The remaining strains are also diploid, with exceptions in some chromosomes: (B) PL448 scaffold5 is present in three copies (two peaks at 33% and 66%), scaffold6 underwent LOH  and end of scaffold2 (2–2.9Mb) is present in three copies (two peaks at 33% and 66% visible beside main peak at 50%); and (C) SZMC21154 scaffold2 from 0 to 2Mb is present in three copies while the remaining 0.9 Mb in two copies (two peaks at 33% and 66% beside main peak at 50%).Distributions of frequencies of read counts at biallelic SNPs in (PDF)Click here for additional data file.S9 FigCandida samples were analyzed using fluorescence-activated cell sorting (FACS): 9 strains of C. metapsilosis, 1 C. parapsilosis, 1 C. orthopsilosis and 1 C. albicans. In the histogram, FITC-A values—fluorescence signal of the fluorochrome fluorescein isothiocyanate—corresponding to DNA content versus cell counts are plotted. Peaks around 50 K and 100 K values of FITC-A account for cells in G1 and G2 phases, respectively. In the table, FITC-A G1 and G2 medians are shown per each of the samples, as well as the ratio between them.Twelve (PDF)Click here for additional data file.S10 Figa / α idiomorphs in six C. metapsilosis strains by four PCR reactions targeting: MTLα1 (150 bp product), MTLα2 (188 bp product), MTL1a (219 bp product) and MTLa2 (216 bp product). MTLα1 and MTLα2 are present in all tested strains. SZMC21154 does not have the MTLa1 not MTLa2 fragments (marked with arrows). B. We have confirmed linearity of MTL a / α idiomorphs in two C. metapsilosis strains (SZMC8092 and SZMC21154) by four PCR reactions flanking introgression ends: P1f ‐ P1rα (932 bp product), P1f –P1ra (900 bp product), P2fα–P2r (1009 bp product) and P2fa–P2r (1076 bp product). MTLα is present in both strains, thus P1f ‐ P1rα and P2fα–P2r yield PCR product in both strains. P1f –P1ra and P2fa–P2r do not give product in SZMC21154, as MTLa is missing in this strain.A. We have confirmed presence of MTL (PDF)Click here for additional data file.S11 FigPhylogenetic trees were reconstructed from matrices consisting of: A) SNPs in the longest (300kb) homozygous regions (51 patterns), B) concatenated chromosomes with incorporated SNP , C1) 13,374 three-state  haplotypes in 1 kb windows , D) 170 multi-state CNVs: 0 for null deletion, 1 for heterozygous deletion, 2 for wild-type (no deletion and duplication), 3 for duplication (3 copies of given locus), 4 for duplication (4 copies of given locus) etc (127 patterns), and E) 8,889 LOH presence / absence profiles (587 patterns). Bootstrap support values are given if lower than 100. Strains are color-coded, accordingly to the place of isolation. Trees were visualised using iTOL .Note, here we consider patterns as phylogenetically informative loci, this is alleles that are shared by more than one strain, but not present in all of them. Maximum Likelihood phylogenetic trees were reconstructed from these alignments using RAxML 7.2.8 using GTRCAT model for all except multi-state matrices and GTRGAMMA model for multi-state matrices.(PDF)Click here for additional data file.S12 FigCandida parapsilosis complex species were aligned using nucmer 3.0.7 : A) C. orthopsilosis 90–125 against C. parapsilosis CDC317, B) C. metapsilosis SZMC8094 against C. parapsilosis CDC317 and C) C. metapsilosis SZMC8094 against C. orthopsilosis 90–125. Forward alignments are colored in red, while reverse alignments in blue. Chromosomes of all three species are syntenic with 8–9 translocations (marked with arrows on the right Y axis) and numerous small inversions.Chromosomes of (PDF)Click here for additional data file.S13 FigC. metapsilosis phylome (http://phylomedb.org/phylome_243) using duptree v1.48 .Parsimony-based species tree were reconstructed based on the topologies of 5,780 gene trees from (PDF)Click here for additional data file.S14 FigDistributions of expected (blue dashed line) and observed LOH blocks from 11 strains for given block size cut-off were plotted. Expected distribution is approximated as E = N * p * q * k, where N is genome size in basepairs, p and q are the probabilities of having a SNP or not, respectively and k is the required size of the block.(PDF)Click here for additional data file.S1 TableC. metapsilosis strain the table provides: its name, available genomic libraries, read length(s), cumulative depth of coverage, k-mer(s) used for assembly, obtained number of contigs, cumulative assembly size, percentage of GC content, number of contigs longer than 1 kb and the cumulative size of these contigs, N50, N90, the cumulative size of gaps and the length of the longest contigs.For each (PDF)Click here for additional data file.S2 TableFor each step of heterozygous assembly the table provides: name, result file name(s), number of contigs, cumulative assembly size, percentage of GC content, number of contigs longer than 1 kb and the cumulative size of these contigs, N50, N90, the cumulative size of gaps and the length of the longest contigs.(PDF)Click here for additional data file.S3 TableC. metapsilosis is diploid, thus wild-type ploidy of 2 is expected. Chromosomes with deviated ploidy are denoted in red (putative duplication) or blue (putative deletion). Note that rDNA cluster is placed in scaffold5, which therefore shows a larger ploidy in all strains. This, however, does not preclude us to detect triploidy of scaffold5 in PL448. Variability in mitochondrial chromosome (scaffold10) copy number is likely reflecting differences in sample preparation and not true biological variability.Estimated ploidy of all chromosomes/scaffolds is provided for all analysed strains. (PDF)Click here for additional data file.S4 TableFor each event duplication and deletion the table lists: its genomic coordinate and size, number of affected strains and their names; estimations of ploidy for these strains in this region; information whether deletion appears as null in at least one strain; affected genes and their functions. For simplicity, several functional classes are provided with a specific color background. Some selected CNVs among those longer than >5kb are annotated in (PDF)Click here for additional data file.S5 TableC. parapsilosis species complex are given. For each cluster, the table lists: cluster ID, total number of genes in the cluster, number of members and their IDs for C. metapsilosis, C. orthopsilosis and C. parapsilosis; functional annotations for cluster members; difference in number of members between C. metapsilosis, C. parapsilosis and C. orthopsilosis. For simplicity, several functional classes are provided with color background.Clusters of orthologous genes that differ in the number of members between (PDF)Click here for additional data file.S6 TableFor each species, the table lists: NCBI Taxonomy species identifier, species name, PhylomeDB proteome code, genome source and date when the genome was downloaded.(PDF)Click here for additional data file.S1 FileFor each chromosome we have plotted: i) coding genes for +/- strand (grey bars) and GC-content in 1kb windows (blue plot) in the bottom track and log2 of observed vs expected value in 1kb windows for depth of coverage (blue) in the top fourteen tracks. In addition, loss of heterozygosity (LOH) regions have been marked in grey, if the same genotype as reference was kept (hapA), and orange, if alternative genotype was kept (hapB). Four replicas  were analysed for PL429. C. metapsilosis genome is a mixture of heterozygous (light grey), haplotype B (dark grey) and haplotype A (orange) regions. We suspect all analysed strains originate from single hybridisation event, as most of LOH events are shared by all strains. Examples of large LOH, duplications and deletions have been annotated ie. rDNA cluster (scaffold5), scaffold5 triploidy in PL448, partial scaffold2 triploidy in SZMC21154 and PL448, and complete LOH in scaffold6 in PL448. For the sake of simplicity, only some selected duplications and deletions among those longer than 5Kb are represented (annotated as such in Supplementary (PDF)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Brotogeris versicolurus), through release of infected confiscated individuals. We developed two mathematical models that describe ND transmission and the influence of illegal harvest in a homogeneous (model 1) and age-structured population of parakeets (model 2). Infection transmission dynamics and harvest were consistent for all individuals in model 1, which rendered it mathematically more tractable compared to the more complex, age-structured model 2 that separated the host population into juveniles and adults. We evaluated the interaction of ND transmission and harvest through changes in the basic reproduction number (R0) and short-term host population dynamics. Our findings demonstrated that ND introduction would likely provoke considerable disease-related mortality, up to 24% population decline in two years, but high harvest rates would dampen the magnitude of the outbreak. Model 2 produced moderate differences in disease dynamics compared to model 1 , but highlighted the importance of adult disease dynamics in diminishing the epidemic potential. Therefore, we suggest that future studies should use a more realistic, age-structured model. Finally, for the presumptive risk that illegal trade of white-winged parakeets could introduce ND into wild populations, our results suggest that while high harvest rates may have a protective effect on the population by reducing virus transmission, the combined effects of high harvest and disease-induced mortality may threaten population survival. These results capture the complexity and consequences of the interaction between ND transmission and harvest in a wild parrot population and highlight the importance of preventing illegal trade.Illegal wildlife-pet trade can threaten wildlife populations directly from overharvest, but also indirectly as a pathway for introduction of infectious diseases. This study evaluated consequences of a hypothetical introduction of Newcastle disease (ND) into a wild population of Peru’s most trafficked psittacine, the white-winged parakeet ( Theics see: –22.Illegal trade in wild-caught birds as pets is common in many regions of the world including southeast Asia ,24, AfriBrotogeris versicolurus) [Peru has among the highest parrot diversities in the world (n = 53 species)  and a locolurus) ,50. Threcolurus) .The white-winged parakeet is a small, non-threatened, highly gregarious species that is common throughout most of its Amazonian range ,53. PeruNewcastle disease is a highly infectious and fatal viral disease caused by avian paramyxovirus serotype-1 that affects many avian species including parrots and poultry . Large eThe ND virus spreads horizontally between healthy and infected birds through direct contact with bodily secretions from infected birds . Crowdedcriollo) chickens and fighting cocks , thus providing the opportunity for cross-species ND transmission. Authorities frequently confiscate white-winged parakeets and immediately release individuals into the wild [White-winged parakeets are susceptible to ND ,74. Duris , with 3s  at different harvest rates [R0 is the average expected number of secondary infections produced by one typical infectious individual introduced into a fully susceptible host population [R0 ≥ 1) or fades out (R0 < 1).Infectious disease mathematical modeling is a useful tool for conservationists and epidemiologists to evaluate potential synergistic effects of illegal trade and disease on wildlife populations and to compare mitigating strategies ,92. The st rates . R0 is tpulation , and is Under certain density-dependent pathogen transmission conditions, harvest can decrease transmission and is the reason culling can be effective to prevent disease spread ,95. In oR0 and disease-related mortality were assessed using a combination of analytical and numerical approaches. We conducted sensitivity and scenario analyses to evaluate the robustness of the models’ results in the presence of uncertainty to individual parameters. Lastly, we discussed the limitations and implications of our results, including the conservation relevance of illegal trade and ND emergence in native populations of white-winged parakeets.We hypothesized that (1) introducing ND into a susceptible population of white-winged parakeets would result in an outbreak with considerable mortality and (2) increasing harvest would lower disease transmission and the magnitude of the outbreak. To evaluate these hypotheses, we developed two continuous-time, mathematical models to assess whether predictions about ND dynamics would differ between a simplified model with a homogeneous bird population and a presumably more realistic, but less tractable, model with an age-structured host population. Both models were considered under different harvest scenarios and Because ND typically results in severe but short-lived epidemics in avian species, we focused our attention on short-term infection dynamics without including density-dependent responses from the host population to the disease-induced mortality . However, given the possibility of long-term chronic infections in parrots , we did SEIAICR model assumed no differences in the infection—transmission dynamics and harvest among age groups. The host population was divided into susceptible (S), exposed (E), acutely-infected (IA), chronically-infected (IC), and recovered (R) states [IA and IC) because experimental evidence suggested two levels of severity of clinical signs, which we used as a proxy for viral shedding [hb = 1%), which we assumed was the current harvest rate of white-winged parakeets in Peru, based on the fact that this species is highly harvested but the population size remains stable [h1) on R0 and host population size.In model 1 , we desc) states . We inclshedding ,99. In ms stable . FurtherOur mathematical framework started with the following assumptions:The host population was a single, free-mixing population and individual parakeets were equally likely to encounter an infected individual. This was a realistic assumption given the gregarious nature of white-winged parakeets and the large number of individuals at communal roost sites, up to 700 to 1,000 individuals ,101.The host population was stable under initial conditions, closed to immigration and emigration, and parakeet natality and mortality were not under the influence of density dependence. While these conditions may not always hold true, they were useful simplifying assumptions that helped isolate the evaluation of harvest and ND dynamics.Both sexes were equally affected by ND virus .At hatching, chicks were susceptible to ND virus. There is limited evidence that psittacine chicks can receive maternal antibodies through the egg ; howeverTransmission of ND was density-dependent where the number of contacts per unit time was proportional to the number of individuals in the population . Densityt = 0) susceptible population, S(0), was set equal to the total population, N(0), minus one individual, which represented an acutely-infectious parakeet introduced into the population, IA(0) = 1 and IAa(0) = 1 for models 1 and 2, respectively. All remaining stages were set to zero. Model simulations and analyses were conducted in Matlab R2015a ; see https://github.com/NDCcodes/Daut_ND-matlab-codes/tree/master or We used a system of ordinary differential equations (ODEs) to describe transmission of ND in white-winged parakeets for each model (Eqs N(d+h) and N changes over time as N(t) = S(t)+E(t)+IA(t)+IC(t)+R(t)Where Λ = N changes over time as N(t) = Nj(t)+Na(t) where Nj(t) = Sj(t)+Ej(t)+IAj(t)+ICj(t)+Rj(t) and Na(t) = Sa(t)+Ea(t)+IAa(t)+ICa(t)+Ra(t)Juvenile:Adult:R0 for our systems of ODEs, we used the next-generation method (NGM) [R0. For model 1 the derived expression for R0 (Eq A1) was:R0A and R0C represent the average number of secondary infections resulting from interactions between susceptible and acutely- and chronically-infected hosts, respectively. For model 2 the derived expression for R0 (Eq A2) was:R01 and R02 represent the average number of secondary juvenile or adult infections, respectively, produced by introduction of one exposed juvenile Ej during its entire infectious period. The terms R03 and R04 represent the average number of secondary juvenile or adult infections produced by introduction of one exposed adult Ea during its entire infectious period, respectively.To calculate od (NGM) . See S2 Parameter notations, definitions, mean values, distributions for sensitivity analyses, and sources of information are described below and in N(0) = 200), a commonly reported flock size [Ʌ) equal to the sum of natural mortality (d) and the current (baseline) harvest (hb) to maintain a stable population in absence of additional harvest (hl), so that Ʌ = N(d+hb) [Dd = 5 years) from captive white-winged parakeets, so that d = 1/Dd [hb = 0.01/365).We defined the initial population of parakeets as  . Daily nd = 1/Dd . Baselinβ) for ND in psittacines have not been published; therefore, we adapted a transmission probability published for backyard poultry that was considered to reasonably represent ND dynamics in wild white-winged parakeets. In a mathematical model describing density-dependent ND dynamics in a backyard chicken flock, Johnston [βA = (3/14)200 individual-1 day-1 (Ir = 10%) of the acute transmission rate, so that βC = βAIr.Transmission coefficients (Johnston  estimate-1 day-1 . ChronicE), acutely-infected (IA), chronically-infected (IC), and recovered (R) stages were used as the daily rates α, δ, γ, and ν, respectively (α = 1/5.5) [δ = 1/30. Although some parrots continued to chronically shed for over a year, on average individuals had stopped shedding by 39 days post infection [γ = 1/39 and was consistent with shedding duration observed in other avian species [ν = 1/243 [According to the standard assumption of exponentially-distributed periods of infection , the recectively ; Fig 1. = 1/5.5) . We base= 1/5.5)  where innfection , which r species ,128,129. species  in exper = 1/243 .p1) of newly infected white-winged parakeets would become acutely infected, and 1–p1 would become chronically infected (p2), while the remainder (1–p2) would become chronically infected.According to ND infection rates in psittacines in captive and experimental conditions, we assumed that all effectively contacted white-winged parakeets would become either acutely or chronically infected, with acutely-infected parakeets shedding more ND virus ,72,130. infected . After aμA = 0.25/30 , so that N(0) = Nj + Na. For instance, when Dd = 5 years and N(0) = 200, Nj = 16 (8%) and Na = 184 (92%) (DΩ = 135 days (4.5 months), meaning that the maturation rate was Ω = 1/135.We set the initial population size of the two age classes according to their distribution at the disease-free equilibrium with life expectancy (84 (92%) . We estiSj(t) = Sj(0)e(–dj*t) so that 60% of the population remained after the juvenile period. The daily natural mortality was then estimated as dj = 1/(–DΩ/ln(0.6)) = 1/264. Adult daily natural mortality da was calculated from the difference between the natural mortality already defined for model 1 (d) and the juvenile mortality (dj), so that da = 1/(1/d–1/dj) (h) to represent adults (ha = 0.6h) and 40% juveniles (hj = 0.4h).We fixed juvenile mortality at 40% during the 135-day long juvenile period ,117. The/d–1/dj) . Accordiβ) for ND in juvenile or adult psittacines have not been published; however, acutely-infected juvenile chickens, pigeons, and other species were considered more infectious than adults [cβA) to represent a 25% increase for acutely-infected juveniles over the baseline rate of transmission (βA) in model 1, so that βAj = (1+cβA)βA, and a 25% decrease for adult transmission below baseline so that βAa = (1–cβA)βA (βC) for juveniles and adults as in model 1. The transition rates for leaving infectious states, α, δ, γ, and ν, used in model 2 were previously defined for model 1 βA ; Fig 2.  model 1 .p1j = 0.75), while 1–p1j would become chronically infected  versus becoming chronically infected was defined as in model 1. We used an acute-mortality factor (cm) to estimate a ± 25% age-related difference so that disease-related mortality for acutely-infected juveniles was μAj = (1+cm)μA and for adults, μAa = (1–cm)μA. Little is known about age-related mortality from chronic ND infection. We assumed that little difference existed among juveniles and adults and, therefore, used the parameter μC from model 1 for both.In natural outbreaks and experimental studies, juvenile birds frequently disproportionately suffered acute clinical signs and high mortality following ND exposure compared to adults ,126,127.infected ; Fig 2. Dd) is unknown in the wild, we conducted a scenario analysis to compare the baseline scenario (Dd = 5 years) with two extreme scenarios (Dd = 2 and Dd = 9 years) for both models. Also, we evaluated the effect of additional harvest (hl) at 2, 5, and 10% along with the 1% annual baseline (hb) harvest  through two scenarios for each model. In the first scenario, population recruitment was fully compensated to include baseline and additional harvest to maintain a stable population in absence of infection, so that Ʌ = N(d+h) in model 1 and Ʌ2 = N(dj+hj+Ω)(da+ha)/(da+ha+Ω) in model 2 (Ʌu = N(d+hb) in model 1 and Λ2u = N(dj+hbj+Ω)(da+hba)/(da+hba+Ω) in model 2, where hbj = 0.4hb and hba = 0.6hb. These two scenarios provided a means to evaluate the interaction between harvest and disease under two different host population reproductive responses to additional harvest .Because life expectancy  and (2) ODE models  was used to calculate and test correlation between each uncertain parameter and R0  or endemic (R0 ≥ 1) states, we used the parameter values and the associated R0 estimates from 10,000 iterations per scenario to construct classification trees—binary decision trees that identify the most influential parameters in predicting disease-free or endemic conditions for our homogeneous and age-structured populations [Dd) and additional harvest (h1), for a total of 120,000 data points (iterations) to determine whether these two, and other, model parameters were important predictors of disease-free or endemic states. Classification trees were built using the rpart package in R software [Spearman’s correlation coefficient . We then varied the age-structured parameters  one at a time to evaluate their individual influence on R0. This provided a method to compare results between the two models and to identify which parameters made the biggest difference in R0 estimates.In a separate analysis, we compared differences in R0 estimates without additional harvest were 3.63 and 2.66 for models 1 and 2, respectively  were slightly wider in model 1 compared to model 2 . The baseline outbreak dynamics peaked at approximately 120 days for the homogeneous population of white-winged parakeets  = 135; N(730) = 153) when age structure was considered (As shown in both panels in N = 200) so that we could evaluate the effects of harvest and disease-related mortality on population decline without the influence of the uncertain initial population size, the width of the 5th and 95th percentiles decreased by over half for both models  was the most influential (positively-correlated) parameter in determining R0 for both homogeneous and age-structured populations of white-winged parakeets (R0. The probability of acute disease-related death (pdA) was negatively correlated with R0 in both models, but had a much stronger influence in model 2  and adult (μAa) parakeets, both of which were partially derived from pdA (R0 in model 2. The probability of chronic disease-related death (pdc) was likewise negatively correlated with R0, but only significant in model 2.The duration of the acute-infectious stage  and additional harvest (h1) were evaluated simultaneously. Parameters Dd and h1 were not identified as influential in predicting disease-free or endemic conditions. As with correlation analyses, the most optimal classification trees identified the duration of the acute-infectious stage (Dδ) as the most important factor determining whether the infection would persist or undergo fade-out, indicated by its position closest to the root of the tree (R0 ≥ 1) were predicted to occur under two scenarios in model 1: (1) when Dδ ≥ 11 days, and (2) when Dδ < 11 days, but with a duration of chronic-infectious stage Dγ ≥ 48 days, and a transmission rate for chronically-infected parakeets βc ≥ 0.0001071 . Cross-validation indicated that the predictive error rates were low and similar for models 1 and 2 .In the correlation analyses, parameters were evaluated individually; in the multivariable classification trees analyses all uncertain parameters as well as life expectancy (the tree . By foll.0001071 . In mode.0001071 . As withcβA), which increased or decreased the transmission rate by 25% for acutely-infected juveniles and adults, respectively, made the biggest difference in R0 between the two models. Specifically, lowering the transmission rate for acutely-infected adult parakeets (βAa) decreased R0 by 21% from 3.63 in model 1 to 2.88 in model 2. Lowering the probability of acute adult infection (p1a) in model 2 compared to the homogenous value (p1) in model 1 decreased R0 by 15%. The acute mortality factor (cm), which lowered the disease-related mortality rate for acutely-infected adult parakeets (μAa), resulted in increasing R0 slightly by 4% compared to model 1. The corresponding increase in juvenile parameter values associated with cβA, p1j and cm, in model 2 minimally influenced R0 compared to model 1.By directly comparing models 1 and 2 and the age-related parameters that differed between the two, we determined that the age-dependent acute transmission factor  in susceptible populations of white-winged parakeets with roughly 75% probability  would provoke > 75% population decline in 20 years without a density-dependent response in fecundity . While wR0 estimates  in model 2  and, to a lesser degree, a lower probability of acute adult infection. Juvenile parakeets, despite being more infectious than adults, made little impact on R0 estimates. This was due, in part, to the fact that 40% of juveniles were removed by natural mortality from the population by day 135, and the remaining would then “mature” to adults  slightly increased R0 compared to model 1. This suggested that maintaining infected adults for longer time in the population , would exacerbate an ND outbreak even though adults were less infectious compared to juveniles.Comparison of models 1 and 2 demonstrated that the lower o adults . Adult po adults , naturalR0 predictions between models 1 and 2  were uncertain and should be prioritized for future investigation, especially the transmission rate and probability for acutely-infected adult parakeets. However, because model 2 more closely reflects the biological system, it is reasonable to assume that it also more accurately predicts ND dynamics. By extension, it is reasonable to conclude that model 1 overestimated disease transmission and the magnitude of the ND outbreak. In a similar situation, Brooks-Pollock et al. [R0 estimates and the effort required for disease control.We should emphasize that the parameters responsible for the differences in k et al.  found thR0, meaning that higher uncompensated harvest increased the probability of ND fadeout  caused R0 to remain high in both models (> 2.5), indicating that an influx of susceptible individuals  may help sustain higher potential for an ND outbreak (R0 estimates with compensated and uncompensated harvest largely overlapped (results not shown), largely because of the wide uniform distributions used in our models , had a modest dampening influence on  R0 < 1; . Althoug R0 < 1; , this inoutbreak ; howeverr models , the infHigher harvest rates produced slower population decline in the age-structured population compared to the homogeneous population . This dahb) conservatively and even compensated for it with natality (h1) was conservatively low. In model 2, we fixed the proportion of juvenile (40%) and adult (60%) harvest as the average of what was recorded in market surveys throughout the year. In reality, we know that there are seasonal harvest differences, which would likely influence ND dynamics as they do for other diseases [R0 was 20% higher, but the population decline at two years post ND introduction was 12% less compared to the opposite proportions .Overall, higher harvest rates had minimal effect on ND dynamics  and popunatality . It is ldiseases ,154. ForDδ) and chronic (Dγ) infectious stages were most influential in determining R0, as identified in both the univariate correlation , is one of the primary goals for improving vaccination programs [pdA) and R0 in both models  and chronic (Dγ) infectious stages, the most optimal classification trees identified the transmission rate for chronically-infected parakeets (βc) as influencing R0, rather than the transmission rate for acutely-infected parakeets (βa) as in our correlation analysis. When the interaction of transmission rates was evaluated along with other parameters, it became clear that the rate of virus transmission from chronically-infected birds (βc) to susceptible individuals would become critical in determining whether ND would die out or persist, specifically when chronic infection period (Dγ) lasted longer than approximately 47 days Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Campylobacter jejuni is a frequent cause of human bacterial gastrointestinal foodborne disease worldwide. Antibiotic resistance in this species is of public health concern. The draft genome sequences of three multiantibiotic-resistant C. jejuni strains  isolated from poultry at retail outlets in Malaysia are presented here. Campylobacter jejuni is one of the leading causes of bacterial gastrointestinal foodborne disease worldwide . The draft genome of C. jejuni 2865 was assembled into 69 contigs, with a 30.2% G+C content and an accumulated length of 1,821,463 bp . The draft genome of C. jejuni 2868 was assembled into 111 contigs, with a 30.3% G+C content and an accumulated length of 1,742,310 bp . The draft genome of C. jejuni 2871 was assembled into 31 contigs, with a 30.4% G+C content and an accumulated length of 1,654,937 bp .Three Malaysia , were seC. jejuni strain 2865, while 1,701 CDSs, 4 rRNAs, and 40 tRNAs were annotated for C. jejuni strain 2868, and 1,650 CDSs, 3 rRNAs, and 40 tRNAs were annotated for C. jejuni strain 2871.Using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST), 1,811 coding sequences (CDSs), 3 rRNAs, and 40 tRNAs were annotated for cadF and peb) (flgE2 and flaAB)  (–csrA) (tet(O) gene, are present in all three strains but absent in reference strains, such as C. jejuni NCTC 11168. The presence of these genes might affect the ability of these three strains to form biofilms.Genes related to biofilm formation processes, such as adhesion (and peb) , 12, motd flaAB) , 14, capnd waaF) –17, and  (–csrA) , are preC. jejuni strains 2865, 2868, and 2871 have been deposited at DDBJ/EMBL/GenBank under the accession numbers LLWL00000000, LLWM00000000, and LLWN00000000, respectively. The versions described in this paper are versions LLWL00000000, LLWM00000000, and LLWN00000000.The whole-genome shotgun projects of


In this study, the sequences of vp4, vp6 and vp7 were used to analyse sequence variation, phylogenetic relationships and genotypes in twenty five GCRV isolates. The results indicated these twenty five GCRV isolates should be attributed to four genotypes. And there were no obvious characteristics in the geographical distribution of GCRV genotype. The study should provide the exact foundation for developing more effective prevention strategies of grass carp haemorrhagic disease.Grass carp reovirus (GCRV) is a causative agent of haemorrhagic disease in grass carp that drastically affects grass carp aquaculture. Here we report a novel GCRV isolate isolated from sick grass carp that induces obvious cytopathic effect in CIK cells and name it as GCRV096. A large number of GCRV 096 viral particles were found in the infected CIK cells by electron microscope. The shape, size and the arrangement of this virus were similar to those of grass carp reovirus. With the primers designed according to GCRV 873 genome sequences, specific bands were amplified from sick grass carp and the infected CIK cells. The homology rates among  Aquareovirus genus and the Reoviridae family, can cause serious haemorrhagic disease in grass carp (Chen and Jiang Grass carp reovirus (GCRV), which is known as a member of the Aquareovirus C species. The genome of GCRV is known to consist of 11 segments of dsRNA contained in a core surrounded with a double-layered icosahedral capsid  were purchased from shenzhen inspection and quarantine bureau in China. CIK is GCRV sensitive cell and are widely used in the related study of GCRV , the GCRV 096 genome RNA was extracted from purified GCRV 096 virus. The cDNA of GCRV 096 genome RNA was acquired with RT-PCR kit  using the random primers and M-MLV reverse transcriptase.vp4, vp6 and vp7 gene amplification were designed based on homologous sequence in GCRV 873: 5″-CACTTCGCACTCTCTCTACAATG-3′ and 5′-AGTACGACACTTCCCGCCGTT-3′, 5′-TGTGATGGCACAGCGTCAG-3′ and 5′-GTTAGA CGAACATCGCCTGC-′3, 5′-TCACCACGATGCCACTTCAC-3′ and 5′-CGGTGCTTAATCGGATGGCT-3′, respectively. Primers were also designed based on homologous sequence from GCRV GD108 and GCRV HZ08 for vp4, vp6 and vp7 gene: 5′-ACTTACGGCCACTATCATGG-3′ and 5′-TCGGTGTACACGACCTAAG-3′, 5′-CTTTGAGTCGACGCACGTAT-3′ and 5′-CCGTCGGGTGGATTAGGTC-3′, 5′-TCTACTGCCAAGATGGCCAC-3′ and 5′-GCACGCACCTTACTTACAGCA-3′. The PCR cycling conditions were an initial denaturation at 95°C for 3 min followed by 30 cycles consisting of 94°C for 30 s, 55°C for 60 s and 72°C for 70 s, and a final extension step of 30 min at 72°C. The composition of the PCR system (50 μl) includes 33 μl sterile water, 3 μl dNTP (each is 2.5 mmol/L), 10 pmol/L primer for 2 μl each, 10 × buffer for 5 μl (containing Mg++), DNA for 100 ng and Taq polymerase for 0.25 μl (5 U/μl). The aimed genes were purified using Gel Extraction Kit  from gelose gel and connected with pMD18-T vector at 16°C, then transformed to DH5α E.coli. The recombined plasmid was verified by sequencing.According to the genome sequence of GCRV 873, primers for GCRV 096 vp4, vp6 and vp7 genes were aligned by using the Clustal V method in DNAstar software. Subsequently, the alignment was manually adjusted. Variable sites, information sites, genetic distances, and homologic rates of segments were calculated with MEGA5.1 .vp4, vp6 and vp7 genes in the these GCRV isolates contain 184, 447 and 375 informative sites, respectively. Table vp4, vp6 and vp7 genes, respectively. Based on the data shown in Table vp4, vp6 and vp7 genes among GCRV 096, GCRV 873, GCRV 875, GCRV JX01, GCRV 876 and GCRV 991 or AGCRV and AGVRV PB01-155 were small, and their homologous rates were high. Also, genetic distances among GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were small, with elevated homologous rates. Other genetic distances were far and the genetic identities were small.vp4, vp6 and vp7 gene from ModelTest3.7 (Posada and Crandall Simulation results of the GCRV evolutionary model based on vp4, vp6 and vp7 genes of GCRV in this article are basically coincident. According to evolutionary simulation results, there is the UPGMA tree constructed based on vp4 gene in Figure Topological structures of constructed phylogenetic trees, based on vp6 gene. On the MP tree, the cluster on the top consisted of GCRV 106, GCRV HeNan988, GCRV HuNan794, GCRV 918, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV GD108 and GCRV HZ08. The second cluster was GCRV 104. The next cluster was AGCRV PB01-155 and AGCRV. The last cluster contained GCRV 096, GCRV 875, GCRV 876, GCRV 991 and GCRV 873.In Figure vp7 gene. The cluster on the top of this tree consisted of GCRV 096, GCRV 875, GCRV 876, GCRV JX01, GCRV 991 and GCRV 873. The second cluster was GCRV GD108, GCRV HZ08 and GCRV JX02. The last cluster contained AGCRV PB01-155 and AGCRV. The phylogenetic relationships of GCRV096, GCRV 991, GCRV 876, GCRV 873, GCRV JX01, and GCRV 875 or GCRV HZ08, GCRV JX02, and GCRV GD108 are relatively close.In Figure vp4 gene; 57.1% and 80.0% in vp6 gene; 77.3% and 89.3% in vp7 gene.By analysing variable sites, we found the ratios of variation sites located on the third condon and transitions were respectively 71.6% and 82.1% in Amongst all aquareovirus isolates, GCRV is one of the most pathogenic agents (Fang et al. vp4, vp6 and vp7 gene encode major outer capsid proteins and consist of many variable sites (Rangel et al. vp4, vp6 and vp7 gene could be used for GCRV genotyping.In order to analyse the difference among GCRV isolates as well as their evolutionary relatiohship, it is necessary to genotyping. Currently, uniform criteria in place for virus genotyping are still unavailable. In hepatitis C virus, a more than 30% nucleotide sequence divergence between genotypes is generally considered standard Simmonds . The genThe genetic distances among GCRV 096, GCRV JX01, GCRV 873, GCRV 875, GCRV 876 and GCRV 991 were small with high homologous rates. Furthermore, these isolates clustered together into one cluster on constructed phylogenetic trees. These results present that GCRV 096, GCRV JX01, GCRV 873, GCRV 875, GCRV 876 and GCRV 991 are attributed to the same genotype, i.e. genotype І. Genetic distances between AGCRV PB01-155 and AGVRV were small and their homologous rates were also high. On phylogenetic trees, AGCRV and AGCRV PB01-155 separately clustered into one cluster. These results indicate that AGCRV and AGCRV PB01-155 are attributed to a new genotype, i.e. genotype II. Genetic distances among GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were extremely small with especially high homologous rates. Furthermore, these isolates clustered together into one cluster on phylogenetic trees. GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were attributed to another new genotype, i.e. genotype III. In contrast, genetic distances between GCRV 104 and other GCRV isolates were large, and their homologous rates were small. On the phylogenetic tree (Figure The genotyping results obtained are consistent with previous research conclusions. The study of Wang indicated there were different genotypes of GCRV in China (Wang et al. vp4, vp6 and vp7 gene were high.The comparative analysis of the geographic location Table  of colleHemorragic disease of grass carp outbreaks seriously in China. Many isolates of grass carp reovirus have been discovered while new isolates are being isolated constantly. The systematic difference comparison of the different GCRV isolates has not been reported. In this study, we have verified the diference among various GCRV genotypes. GCRV genotyping has important significance to diagnosis and treatment in hemorrhagic disease of grass carp, especially to vaccine development. Comparison of different GCRV isolates and genotyping are helpful to further our understanding in GCRV genetic variation and evolution and the development of more effective preventative strategies against GCRV.This study provides a foundation for revealing differences among GCRV isolates. Simultaneously, it is significant for the further research on genetically engineered vaccines against grass carp haemorrhagic disease and grass carp breeding for disease resistance.


Cryptosporidium and Giardia in faecal samples from various animals in nine water reservoir areas over a period of 4 years (July 2011 to November 2015).In a long-term program to monitor pathogens in water catchments serving the City of Melbourne in the State of Victoria in Australia, we detected and genetically characterised SSU) and 60 kDa glycoprotein (gp60) genes for Cryptosporidium, and triose-phosphate isomerase (tpi) gene for Giardia.This work was conducted using PCR-based single-strand conformation polymorphism (SSCP) and phylogenetic analyses of portions of the small subunit of ribosomal RNA ; 25 distinct sequence types were defined for pSSU, and six for gp60 which represented C. hominis (genotype Ib - subgenotype IbA10G2), C. cuniculus , and C. canis, C. fayeri, C. macropodum, C. parvum, C. ryanae, Cryptosporidium sp. “duck” genotype, C. suis and C. ubiquitum as well as 12 novel SSU sequence types. The prevalence of Giardia was 0.31 % ; all three distinct tpi sequence types defined represented assemblage A of G. duodenalis.The prevalence of Cryptosporidium and Giardia, respectively, from humans. Novel genotypes of Cryptosporidium and Giardia were recorded for SSU (n = 12), gp60 (n = 4) and tpi (n = 1); the zoonotic potential of these novel genotypes is presently unknown. Future work will continue to monitor the prevalence of Cryptosporidium and Giardia genotypes in animals in these catchments, and expand investigations to humans. Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. KU531647–KU531718.Of the 34 sequence types (genotypes) characterized here, five and one have been recorded previously for The online version of this article (doi:10.1186/s13071-016-1607-1) contains supplementary material, which is available to authorized users. Cryptosporidium and Giardia are highly significant [Cryptosporidium and Giardia are unique in that very small numbers of infective stages  can cause disease in humans [One of the toughest challenges facing the world’s supply of clean drinking water is contamination from faeces and soil , 2. Diarnificant , 4–8. Crn humans , 10 and n humans . An examn humans , which aCryptosporidium and Giardia in faecal samples from various mammals and birds in the Melbourne’s catchments [SSU and gp60) in the small subunit (SSU) of ribosomal RNA and 60-kDa glycoprotein (gp60) genes to detect and characterise Cryptosporidium, and another locus (tpi) in the triose-phosphate isomerase (tpi) gene to identify and classify Giardia [Cryptosporidium and Giardia were detected in 2.8 and 3.4 % of all 2009 samples tested, respectively, and 35 previously undescribed genotypes were reported [Melbourne  is one of the few cities in the world that receives largely unfiltered drinking water from protected wilderness catchment areas. The management of Melbourne’s ten main water catchment areas includes restricted access for humans, long water retention times and an intense program of testing and monitoring for pathogens in source water. These catchments represent habitat for native and feral animals, such that the monitoring of zoonotic pathogens is central to management and the prevention of outbreaks of waterborne disease. In 2008, we initiated a program to monitor tchments . To do ttchments –16), com Giardia . In totareported . In spitCryptosporidium and Giardia from native and introduced animals in Melbourne’s water catchments, in order to continually assess the prevalence and diversity of Cryptosporidium and Giardia taxa, evaluate their host affiliations, geographical distributions and zoonotic potential, and support catchment management. In the present article, we describe the results from this 4-year study and discuss the findings in a water industry context.Therefore, from July 2011 to November 2015, we extended our monitoring program, and genetically characterised Greater Melbourne sources its municipal drinking water from ten main water catchment reservoirs. Approximately 80 % of Melbourne’s drinking water is drawn from ‘closed’ catchments in the Yarra Ranges (~85 km east of Melbourne), which cover 157,000 hectares of eucalypt forest, with restricted human and domestic animal access, to minimise the risk of waterborne diseases. The remaining 20 % of Melbourne’s water comes from ‘open’ catchments, in which some farming and human activities are permitted. All water undergoes treatment in accordance with national and international guidelines , 18. TheCanis familiaris (dog), Vulpes vulpes (fox), Felis catus (cat), Dromaius novaehollandiae (emu), Wallabia bicolor , Macropus giganteus (Eastern grey kangaroo), Trichosurus vulpecula (common brushtail possum), Oryctolagus cuniculus (rabbit), Rattus lutreolus (swamp rat), Rattus fuscipes (bush rat) and Vombatus ursinus (common wombat), together with waterbirds, principally the Australian wood duck (Chenonetta jubata), and deer, including Rusa unicolour (sambar), Cervus elaphus (red) and Dama dama , and samples of unknown host origin were collected from nine locations from July 2011 to November 2015 , GV (n = 638), MR (n = 344), OS (n = 622); SV (n = 527), TAR (n = 61), TH (n = 31), UY (n = 714) and YY (n = 601). Scats were identified using a field guide [b gene from faecal DNA using a similar approach to that described by Dalen et al. [A total of 4,256 faecal deposits from ld guide , and hosCryptosporidium, a portion of the SSU gene (~240 bp) was used [gp60 gene (250–350 bp) [Giardia (to the level of assemblage), a portion of the tpi gene (~530 bp) was employed [2 (depending on the locus), 200 μM of each deoxynucleotide triphosphate, 50 pmol of each primer and 1 U of either GoTaq (Promega) or MangoTaq™  DNA polymerase.Genomic DNA samples were subjected to nested PCR-based analyses of three loci. For the specific identification of was used , and gen–350 bp) . For theemployed . PCR wasCryptosporidium, primary amplification of SSU was achieved using primers XF2 (forward: 5'-GGA AGG GTT GTA TTT ATT AGA TAA AG-3') and XR2 (reverse: 5'-AAG GAG TAA GGA ACA ACC TCC A-3') [SSU using the internal primers pSSUf (forward: 5'-AAA GCT CGT AGT TGG ATT TCT GTT-3') and pSSUr (reverse: 5'-ACC TCT GAC TGT TAA ATA CRA ATG C-3') [For CC A-3') , followeTG C-3') . For priCryptosporidium was further characterized using a longer region (~590 bp) of the SSU gene. This region was first PCR-amplified employing primers 18SiCF2 (forward: 5'-GAC ATA TCA TTC AAG TTT CTG ACC-3') and 18SiCR2 (reverse: 5'-CTG AAG GAG TAA GGA ACA ACC-3'), followed by a nested amplification using primers 18SiCF1 (forward: 5'-CCT ATC AGC TTT AGA CGG TAG G-3') and 18SiCR1 (reverse: 5'-TCT AAG AAT TTC ACC TCT GAC TG-3') [For selected samples, C TG-3') . Both amgp60 gene (~1 kb) was first amplified using primers gp15-ATG (forward: 5'-ATG AGA TTG TCG CCT CAT TAT C-3') and gp15-STOP (reverse: 5'-TTA CAA CAC GAA TAA GGC TGC-3') [gp60 using primers gp15-15A (forward: 5'-GCC GTT CCA CTC AGA GGA AC-3') and gp15-15E (reverse: 5'-CCA CAT TAC AAA TGA AGT GCC GC-3') [gp60 utilised the cycling protocol, 94 °C for 5 min , followed by 35 cycles of 94 °C for 30 s (denaturation), 55 °C for 45 s  and 72 °C for 1 min (extension), with a final extension of 72 °C for 10 min. For the amplification of gp60, we employed 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min.The  TGC-3') , followeC GC-3') . PrimaryCryptosporidium was further characterized using a longer region (~850 bp) of the gp60 gene. This region was PCR-amplified using primers AL3531 (forward: 5'-ATA GTC TCC GCT GTA TTC-3') and AL3535 (reverse: 5'-GGA AGG AAC GAT GTA TCT-3'), followed by a nested amplification using primers AL3532 (forward: 5'-TCC GCT GTA TTC TCA GCC-3') and AL3534 (reverse: 5'-GCA GAG GAA CCA GCA TC-3') [For some samples, A TC-3') . For botGiardia, the tpi locus was amplified using primers AL3543 (forward: 5'-AAA TTA TGC CTG CTC GTC G-3') and AL3546 (reverse: 5'-CAA ACC TTT TCC GCA AAC C-3’), followed by the nested amplification of tpi employing primers AL3544 (forward: 5'-CCC TTC ATC GGT GGT AAC TT-3') and AL3545 (reverse: 5'-GTG GCC ACC ACT CCC GTG CC-3') [tpi was achieved employing 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.For G CC-3') . For theSSU and gp60 amplicons  and then subjected to electrophoresis at 74 V at 7.4 °C (constant) for 16 h in a GMA Wide Mini S-2x25 gel in a SEA 2000 rig (Elchrom Scientific AG) using TAE buffer . A control sample (representing a known genotype) was included on each gel to ensure the reproducibility of profiles representing this sample among gels.Single-strand conformation polymorphism (SSCP) analysis was used to scan for sequence variation within and among ns e.g. , 16). In. InSSU aSSU, gp60 (Cryptosporidium) and tpi (Giardia) amplicons were treated with shrimp alkaline phosphatase and exonuclease I , according to the manufacturer’s instructions, and then subjected to bi-directional automated sequencing  using the same primers employed in the secondary PCR. Sequence quality was verified by comparison with corresponding electropherograms using the program Geneious v.8 [Following SSCP-based analysis, selected amplicons representing each distinct electrophoretic profile and all ious v.8 . Sequencious v.8 , and aliious v.8 . SequencSSU data were based on the Akaike Information Criteria (AIC) test in jModeltest v.2.1.7 [SSU (Cryptosporidium) and tpi (Giardia) data, the number of substitutions (Nst) was set at 6, with a gamma-distribution and a proportion of invariable sites. For the separate analyses of gp60 (Cryptosporidium) sequence data, the Nst was set at 6, with an equal rate among sites. Posterior probability (pp) values were calculated by running 2,000,000 generations with four simultaneous tree-building chains. Trees were saved every 100th generation. At the end of each run, the standard deviation of split frequencies was < 0.01, and the potential scale reduction factor approached one. A 50 % majority rule consensus tree for each analysis was constructed based on the final 75 % of trees generated by BI. Analyses were run three times to ensure convergence and insensitivity to priors. Outgroups used in the analyses were Giardia muris for G. duodenalis (tpi), C. hominis for gp60 and C. muris for SSU.Phylogenetic analysis of sequence data was conducted by Bayesian inference (BI) using Monte Carlo Markov Chain (MCMC) analysis in MrBayes v.3.2.3 . The lik v.2.1.7 . For SSUn = 69) produced from 4,256 (1.62 %) faecal DNA samples and identified them to species and/or genotype of Cryptosporidium. A total of 64 samples were characterised by their SSU sequences; 52 were assigned GenBank accession nos.  to the most closely related species of Cryptosporidium in particular clades based on sequence identity:C. fayeri clade:Members of the C. fayeri. Two of 287  and one of 603  samples from kangaroos were test-positive for Cryptosporidium sp. EGK1 (eastern grey kangaroo type 1), two of which were novel genotypes (GenBank accession nos. KU531656 and KU531666). One of 76 (1.32 %) samples from wallabies from catchment CA was a novel genotype that was similar to C. fayeri (KU531658). One of the 287 (0.35 %) samples from kangaroos from catchment YY was a novel genotype similar to Cryptosporidium sp. kangaroo genotype I (KU531694). For the first time, C. fayeri and C. fayeri-like genotypes were reported from wombats.Six of 74  samples from wallabies were test-positive for C. hominis, C. parvum and C. cuniculus clade:Members of the C. hominis. One of 603 (0.17 %) samples from a kangaroo from catchment GV was test-positive for C. parvum. Five of 26 , one of eight  samples and one of 287  samples from rabbits were test-positive for C. cuniculus.One of 536  and one of 408  from deer were test-positive for C. canis clade:Members of the C. canis.One of 52 (1.92 %) samples from emus from catchment CA was test-positive for C. suis clade:Members of the C. suis-like genotype (KU531660).One of 536 (0.19 %) samples from deer from catchment UY was test-positive for the novel C. ubiquitum clade:Members of the C. ubiquitum. Two samples from wombats were test-positive for a novel C. ubiquitum-like genotype.Two of 408 (0.49 %) and one of 247 (0.40 %) samples from deer from catchments OS and CA, respectively, were test-positive for C. macropodum clade:Members of the C. macropodum. Four of 53 (7.55 %) samples from wallabies from catchment OS were test-positive for C. macropodum-like genotypes, three of which were novel . One of 168 (0.60 %) samples from wallabies from catchment SV were test-positive for a C. macropodum-like genotype.Twelve of 603 (1.99 %) and one of 287 (0.35 %) of kangaroos from GV and YY, respectively, were test-positive for Cryptosporidium sp. duck genotype clade:Members of the Two novel genotypes were identified from three of 55 (5.45 %) samples from waterbirds from catchment UY (KU531654 and KU531689).C. ryanae clade:Members of the Cryptosporidium sp. deer genotype from the catchments UY and YY, respectively. One of 536 , two of 408 , two of 247  and one of 250  samples from deer were test-positive for the C. ryanae-like MW4 genotype. Two of 408 (0.49 %) samples from deer from catchment OS were test-positive for the novel genotype C. ryanae-like MW7 (KU531650). Two of 408  and one of 247  samples from deer were test-positive for the C. ryanae-like MW2 genotype.Two of 536 (0.37 %) and one of 250 (0.40 %) samples from deer were test-positive for In the following list, we assign individual taxa identified in the present study (based on the sequencing of gp60 (n = 10) were characterised to the level of subgenotype, and seven of them were assigned GenBank accession nos. (KU531699–KU531705). Based on a comparison with reference sequences from GenBank, six unique gp60 sequence types were characterised as C. hominis (genotype Ib - subgenotype IbA10G2), and C. cuniculus (genotype Vb - subgenotype VbA25 and VbA26) . In addition, Cryptosporidium cuniculus subgenotypes VbA25 and VbA26 were identified in seven samples from rabbits from catchments GV, YY, SV and MR, four of which were novel. Cryptosporidium cuniculus (VbA26) was identified in two samples from kangaroos from catchment YY (cf. [All ten samples test-positive in PCR for gp60 n = 0 were ch YY (cf. ).Fig. 3Rtpi amplicons identified 13 of 4,256 (0.31 %) individual faecal samples to contain Giardia representing the genetic assemblage A of G. duodenalis, based on direct sequence comparisons. From the 13 samples, we defined three distinct sequence types for tpi  of Giardia from deer identified here was novel. Giardia sub-assemblage AIII was identified in nine samples from deer, including five of 536 , two of 408  and two of 18  samples from three different catchments.Sequencing of all Cryptosporidium and/or Giardia have been described from catchment regions in New South Wales [Extending our initial monitoring program , the preth Wales –42, Queeth Wales , Victorith Wales , 33, 44 th Wales .Cryptosporidium and 3.4 % for Giardia [n = 4,256), yet the prevalence of Cryptosporidium and Giardia were 1.62 % and 0.31 %, respectively. Overall, the prevalence of these protists was usually less than that reported previously in New South Wales [Cryptosporidium and Giardia in Melbourne’s catchments over the six-year duration of the project, including animal culls, changing water levels of the reservoirs and the end of a nine-year drought [Giardia in the earlier years , 2,009 faecal samples were collected and tested, resulting in a prevalence of 2.8 % for  Giardia . In contth Wales , 39, 42,th Wales  and Westth Wales . The low drought . One facars (cf. ) was a hCryptosporidium (represented by the GenBank accession nos. KU531647–KU531705), and to the genetic assemblage ‘AI’ and ‘AIII’ of G. duodenalis (accession nos. KU531706–KU531718). Of the recognised species of Cryptosporidium, only C. macropodum, detected here in kangaroos, has not been reported previously from humans. The remaining six potentially zoonotic species  were recorded from deer, emu, kangaroo, rabbit, wallaby and wombat, respectively, and from catchments CA, GV, OS, MR, SV, UY and YY  might suggest a low risk of waterborne transmission to humans. Nonetheless, other species and genotypes of Cryptosporidium may have some zoonotic significance. For example, C. cuniculus, with a prevalence of 0.26 %, was implicated in a zoonotic outbreak of cryptosporidiosis in humans in England in 2008 [Cryptosporidium ubiquitum, detected here in deer in catchments CA and UY, might also be a concern, as it has been proposed to present a potential public health risk due to its broad geographical and host ranges, including humans in industrialised nations [C. canis (0.02 %) and C. fayeri (0.26 %) might also represent minor risks to humans; C. canis has been detected in humans around the world [C. fayeri was detected in a patient in New South Wales suffering from a prolonged gastrointestinal illness [In the present study, we used a PCR-based approach to genetically characterise 82 samples, which were assigned to seven recognised species of vum (see ); these vum see , 53). De. DeCrypt in 2008  and was  in 2008 , 56, and in 2008 . Cryptos nations , 57, 58.he world , and C.  illness .Cryptosporidium to particular clades of species based on sequence identity in SSU, and to genotypes/subgenotypes also based on their gp60 sequence. Within the C. fayeri clade, several novel genotypes characterised from samples from kangaroos were all very similar genetically to the marsupial-specific C. fayeri. Here, we also report the first molecularly characterized Cryptosporidium genotypes from wombats. To date, there is only one record of Cryptosporidium from a wombat [Cryptosporidium of marsupials [Cryptosporidium in multiple surveys [Cryptosporidium and all were genetically very similar to C. fayeri  to be test-positive (by either SSU and/or gp60) for members of this clade compared with our previous study (n = 32) [n = 97) than in the previous study (n = 263) [C. hominis positive (IbA10G2) from a sample from a wallaby from catchment MR was identical in gp60 sequence to a novel C. hominis genotype found in a human in Tasmania  . Considen = 263) . The C. J506839; ), and isrecently . Within  insects , which cand rats , 67. WitCryptosporidium genotypes  as zoonotic agents remains to be proven  and other native and introduced wildlife in Australia, particularly in areas surrounding water catchments, are necessary, not only to determine the significance of various host groups as primary sources, potential reservoirs and amplifiers of Cryptosporidium for transmission to humans, but also to establish the mode(s) of transmission among reservoir animal hosts and how infection is maintained in wild animal populations [Cryptosporidium species infecting wild and domesticated animals not yet studied.The potential role of some ven cf. , 70). Fu. FuCryptulations . The preGiardia, G. duodenalis is responsible for human disease [tpi detected herein represented assemblage A; two have been reported previously (e.g. GenBank accession nos. KU531708 and KU531707)  (cf. ), and on animals , 74, 77,Cryptosporidium and Giardia in animals in key water catchments in Victoria. The genetic analyses indicated that 1.92 % of the 4,256 faecal samples contained Cryptosporidium or Giardia that matched species, genotypes or assemblages with the potential to infect humans. In addition, a number of new sequence records, which did not match any previously published genotypes, were identified. As nothing is known about the zoonotic potential of these new variants of Cryptosporidium and Giardia, future work should establish whether they are found in humans in Australia. Although the focus of the present study was on vast water catchment areas in south-eastern Australia, the research findings and the approach taken have considerable implications for other protected wilderness catchment areas around the world that supply unfiltered drinking water to millions of people.The present study has provided detailed insights into the taxa of gp60, 60 kDa glycoprotein gene; MCMC, Monte Carlo Markov Chain; ORF, open reading frame; pp, posterior probability; SSCP, single-strand confirmation polymorphism; SSU, small subunit of ribosomal RNA; tpi, triose-phosphate isomerase geneAIC, Akaike information criteria; BI, Bayesian inference;