Diversity and molecular epidemiology of Ostreid herpesvirus 1 in farmed Crassostrea gigas in Australia: Geographic clusters and implications for "microvariants" in global mortality events.

Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or "microvariant", i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10-3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Alien Smooth Newts (Lissotriton vulgaris) in Australia Are Infected with Batrachochytrium dendrobatidis but Test Negative for Ranaviruses.

Smooth newts (Lissotriton vulgaris) established recently in Melbourne, Australia. Previously, the population's disease status was unknown. Samples from 34 adults and 78 larvae, collected 2011-16, were tested for two pathogens driving the global amphibian extinction crisis. The fungus Batrachochytrium dendrobatidis was identified (6.3% quantitative PCR positive); ranaviruses were not detected.

The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus.

Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range.

Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being documented across southeastern Australia in New South Wales (NSW), the Australian Capital Territory (ACT), Victoria, and South Australia. We conducted a survey for EHNV between July 2007 and June 2011. The disease occurred in juvenile redfin perch in ACT in December 2008, and in NSW in December 2009 and December 2010. Based on testing 3622 tissue and 492 blood samples collected from fish across southeastern Australia, it was concluded that EHNV was most likely absent from redfin perch outside the endemic area in the upper Murrumbidgee River catchment in the Murray⁻Darling Basin (MDB), and it was not detected in other fish species. The frequency of outbreaks in redfin perch has diminished over time, and there have been no reports since 2012. As the disease is notifiable and a range of fish species are known to be susceptible to EHNV, existing policies to reduce the likelihood of spreading out of the endemic area are justified.

Susceptibility of Australian Redfin Perch Perca fluviatilis Experimentally Challenged with Epizootic Hematopoietic Necrosis Virus (EHNV).

The ranavirus epizootic hematopoietic necrosis virus (EHNV) is endemic to Australia and is listed by the Office International des Epizooties. Clinical outbreaks have only been observed in wild populations of Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and farmed populations of Rainbow Trout Oncorhynchus mykiss. The initial outbreaks of EHNV describe all age-classes of Redfin Perch as being susceptible and can lead to epidemic fish kills. Subsequently, experimental challenge studies using either cohabitation with the virus or injection exposures resulted in mixed susceptibilities across various age-groupings of Redfin Perch. We used an experimental bath challenge model to investigate the susceptibility of Redfin Perch collected from areas with and without a history of EHNV outbreaks. The median survival time for fish from Blowering Dam in New South Wales, a zone with a history of EHNV outbreaks, was 35 d, compared with fish from other areas, which had a median survival between 12 and 28 d postexposure. Redfin Perch from Blowering Dam demonstrated an increased mortality associated with epizootic hematopoietic necrosis up to approximately day 14 after exposure, and then there was a significantly reduced risk of mortality until the end of the trial compared with all other fish. Redfin Perch from Blowering Dam had markedly decreased susceptibility to EHNV, and less than 40% became infected following a bath challenge. In contrast, Redfin Perch from neighboring (e.g., Bethungra Dam and Tarcutta Creek) and distant water bodies (e.g., in Western Australia) with no previous history of EHNVdisplayed moderate to high susceptibility when given a bath challenge. Potential factors for the observed changes in the host-pathogen relationship include intense positive selection pressure for resistant fish following epizootic hematopoietic necrosis outbreaks and subsequent attenuation of the virulence of the virus in resistant fish. Received August 22, 2015; accepted February 13, 2016.

Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus).

The movement of ornamental fish through international trade is a major factor for the transboundary spread of pathogens. In Australia, ornamental fish which may carry dwarf gourami iridovirus (DGIV), a strain of Infectious spleen and kidney necrosis virus (ISKNV), have been identified as a biosecurity risk despite relatively stringent import quarantine measures being applied. In order to gain knowledge of the potential for DGIV to enter Australia, imported ornamental fish were sampled prior to entering quarantine, during quarantine, and post quarantine from wholesalers and aquatic retail outlets in Australia. Samples were tested by quantitative polymerase chain reaction (qPCR) for the presence of megalocytivirus. Farmed and wild ornamental fish were also tested. Megalocytivirus was detected in ten of fourteen species or varieties of ornamental fish. Out of the 2086 imported gourami tested prior to entering quarantine, megalocytivirus was detected in 18.7% of fish and out of the 51 moribund/dead ornamental fish tested during the quarantine period, 68.6% were positive for megalocytivirus. Of fish from Australian wholesalers and aquatic retail outlets 14.5% and 21.9%, respectively, were positive. Out of 365 farmed ornamental fish, ISKNV-like megalocytivirus was detected in 1.1%; these were Platy (Xiphophorus maculatus). Megalocytivirus was not detected in free-living breeding populations of Blue gourami (Trichopodus trichopterus) caught in Queensland. This study showed that imported ornamental fish are vectors for DGIV and it was used to support an import risk analysis completed by the Australian Department of Agriculture. Subsequently, the national biosecurity policy was revised and from 1 March 2016, a health certification is required for susceptible families of fish to be free of this virus prior to importation.

Experimental infection of Australian freshwater fish with epizootic haematopoietic necrosis virus (EHNV).

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray-Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified. Received May 21, 2012; accepted November 1, 2012.

Optimization of Betanodavirus culture and enumeration in striped snakehead fish cells.

An optimized culture method for detection of infection of fish with the Red spotted grouper nervous necrosis virus (RGNNV) genotype of betanodavirus in striped snakehead (SSN-1, Channa striatus) cells is described. Inoculation of fish tissue homogenates at the same time or within 4 hr of seeding the SSN-1 cells was as sensitive as the method recommended by the World Organization for Animal Health, where homogenates were adsorbed onto an established cell monolayer. Such modification halved the time required and the costs of consumables, and reduced the potential for error when processing large numbers of samples. Positive culture results were obtained from 88.3% of 392 fish tissue homogenates in which RGNNV was detected using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay; 99.7% of 943 tissue homogenates, which were qRT-PCR negative, were cell culture negative. Cytopathic effect (CPE) was characterized by large intracytoplasmic vacuoles in 0.1-60% of cells. Detachment of affected cells from the culture surface resulting in progressive disruption of the monolayer occurred in 46.4% of primary cultures and 96.0% of subcultures of positive samples. Identification of CPE that did not disrupt the cell monolayer increased estimates of the 50% tissue culture infective dose (TCID(50)) by 1.07-2.79 logs (95% confidence interval). The predicted mean TCID(50)/ml was 3.3 logs higher when cells were inoculated less than 36 hr after subculture at less than 80% confluence compared to cells inoculated at greater than 80% confluence and more than 36 hr after subculture (P < 0.05).

Discovery of mating in the major African livestock pathogen Trypanosoma congolense.

The protozoan parasite, Trypanosoma congolense, is one of the most economically important pathogens of livestock in Africa and, through its impact on cattle health and productivity, has a significant effect on human health and well being. Despite the importance of this parasite our knowledge of some of the fundamental biological processes is limited. For example, it is unknown whether mating takes place. In this paper we have taken a population genetics based approach to address this question. The availability of genome sequence of the parasite allowed us to identify polymorphic microsatellite markers, which were used to genotype T. congolense isolates from livestock in a discrete geographical area of The Gambia. The data showed a high level of diversity with a large number of distinct genotypes, but a deficit in heterozygotes. Further analysis identified cryptic genetic subdivision into four sub-populations. In one of these, parasite genotypic diversity could only be explained by the occurrence of frequent mating in T. congolense. These data are completely inconsistent with previous suggestions that the parasite expands asexually in the absence of mating. The discovery of mating in this species of trypanosome has significant consequences for the spread of critical traits, such as drug resistance, as well as for fundamental aspects of the biology and epidemiology of this neglected but economically important pathogen.

Genetic analysis of the human infective trypanosome Trypanosoma brucei gambiense: chromosomal segregation, crossing over, and the construction of a genetic map.

BACKGROUND: Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense. RESULTS: The genetic map was determined using 119 microsatellite markers assigned to the 11 megabase chromosomes. The total genetic map length of the linkage groups was 733.1 cM, covering a physical distance of 17.9 megabases with an average map unit size of 24 kilobases/cM. Forty-seven markers in this map were also used in a genetic map of the nonhuman infective T. b. brucei subspecies, permitting comparison of the two maps and showing that synteny is conserved between the two subspecies. CONCLUSION: The genetic linkage map presented here is the first available for the human-infective trypanosome T. b. gambiense. In combination with the genome sequence, this opens up the possibility of using genetic analysis to identify the loci responsible for T. b. gambiense specific traits such as human infectivity as well as comparative studies of parasite field populations.

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates.

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.

The genetic map and comparative analysis with the physical map of Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and contributes to the debilitating disease 'Nagana' in cattle. To date we know little about the genes that determine drug resistance, host specificity, pathogenesis and virulence in these parasites. The availability of the complete genome sequence and the ability of the parasite to undergo genetic exchange have allowed genetic investigations into this parasite and here we report the first genetic map of T.brucei for the genome reference stock TREU 927, comprising of 182 markers and 11 major linkage groups, that correspond to the 11 previously identified chromosomes. The genetic map provides 90% probability of a marker being 11 cM from any given locus. Its comparison to the available physical map has revealed the average physical size of a recombination unit to be 15.6 Kb/cM. The genetic map coupled with the genome sequence and the ability to undertake crosses presents a new approach to identifying genes relevant to the disease and its prevention in this important pathogen through forward genetic analysis and positional cloning.

Allelic segregation and independent assortment in T. brucei crosses: proof that the genetic system is Mendelian and involves meiosis.

The genetic system on Trypanosoma brucei has been analysed by generating large numbers of independent progeny clones from two crosses, one between two cloned isolates of Trypanosoma brucei brucei and one between cloned isolates of T. b. brucei and Trypanosoma brucei gambiense, Type 2. Micro and minisatellite markers (located on each of the 11 megabase housekeeping chromosomes) were identified, that are heterozygous in one or more of the parental strains and the segregation of alleles at each locus was then determined in each of the progeny clones. The results unequivocally show that alleles segregate in the predicted ratios and that alleles at loci on different chromosomes segregate independently. These data provide statistically robust proof that the genetic system is Mendelian and that meiosis occurs. Segregation distortion is observed with the minisatellite locus located on chromosome I of T. b. gambiense Type 2 and neighboring markers, but analysis of markers further along this chromosome did not show distortion leading to the conclusion that this is due to selection acting on one part of this chromosome. The results obtained are discussed in relation to previously proposed models of mating and support the occurrence of meiosis to form haploid gametes that then fuse to form the diploid progeny in a single round of mating.

The sequence and analysis of Trypanosoma brucei chromosome II.

We report here the sequence of chromosome II from Trypanosoma brucei, the causative agent of African sleeping sickness. The 1.2-Mb pairs encode about 470 predicted genes organised in 17 directional clusters on either strand, the largest cluster of which has 92 genes lined up over a 284-kb region. An analysis of the GC skew reveals strand compositional asymmetries that coincide with the distribution of protein-coding genes, suggesting these asymmetries may be the result of transcription-coupled repair on coding versus non-coding strand. A 5-cM genetic map of the chromosome reveals recombinational 'hot' and 'cold' regions, the latter of which is predicted to include the putative centromere. One end of the chromosome consists of a 250-kb region almost exclusively composed of RHS (pseudo)genes that belong to a newly characterised multigene family containing a hot spot of insertion for retroelements. Interspersed with the RHS genes are a few copies of truncated RNA polymerase pseudogenes as well as expression site associated (pseudo)genes (ESAGs) 3 and 4, and 76 bp repeats. These features are reminiscent of a vestigial variant surface glycoprotein (VSG) gene expression site. The other end of the chromosome contains a 30-kb array of VSG genes, the majority of which are pseudogenes, suggesting that this region may be a site for modular de novo construction of VSG gene diversity during transposition/gene conversion events.

The DNA sequence of chromosome I of an African trypanosome: gene content, chromosome organisation, recombination and polymorphism.

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99-100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.