Cherax quadricarinatus Resistant to Chequa iflavirus: A Pilot Study

High mortalities of redclaw crayfish (Cherax quadricarinatus) were reported from northern Queensland farms, mainly attributed to two viruses, Chequa iflavirus and Athtab bunyavirus. From a research population of redclaw crayfish with these pre-existing viral infections, five individuals were found uninfected by Chequa iflavirus but infected with Athtab bunyavirus. A pilot study was designed to examine if progeny crayfish from this cohort were resistant to infections by Chequa iflavirus. Two experiments measured changes in viral load with RT-qPCR. Seven donors, four negative controls and six crayfish injected with a purified virus or saline were used. In Experiment 1, the purified viral inoculum was injected into the crayfish, and they were bled 14 days post-injection (dpi). In Experiment 2, haemolymph containing the viruses was injected into the same crayfish and they were bled at 24 hpi, 48 hpi, 7 dpi and 14 dpi. In Exp. 1, the crayfish cleared Chequa iflavirus infections within 14 dpi, while in Exp. 2, it was within 24 hpi. One mortality was observed, but that crayfish had cleared the virus before dying. The number of copies of Athtab bunyavirus and the weights of the crayfish did not differ significantly (p > 0.05) between the control and injected crayfish. Histology of crayfish all showed that the haemolymph vessels were clear of granulomas, suggesting no bacterial involvement. There was no melanisation in the gill tissue of control crayfish, but it was prominent in virus-injected crayfish. Neither group had haemocytic infiltration of the muscle fibres. Anti-viral immune mechanisms of RNA interference and Cherax quadricarinatus Down Syndrome Cell Adhesion Molecule (DSCAM) are hypothesised to be involved in viral clearance. We conclude that these crayfish were resistant to Chequa iflavirus infections and could be commercially exploited by aquaculturists as a nuclear breeding stock if numbers are increased over time

Disease risk analysis in sea turtles: a baseline study to inform conservation efforts

The impact of a range of different threats has resulted in the listing of six out of seven sea turtle species on the IUCN Red List of endangered species. Disease risk analysis (DRA) tools are designed to provide objective, repeatable and documented assessment of the disease risks for a population and measures to reduce these risks through management options. To the best of our knowledge, DRAs have not previously been published for sea turtles, although disease is reported to contribute to sea turtle population decline. Here, a comprehensive list of health hazards is provided for all seven species of sea turtles. The possible risk these hazards pose to the health of sea turtles were assessed and “One Health” aspects of interacting with sea turtles were also investigated. The risk assessment was undertaken in collaboration with more than 30 experts in the field including veterinarians, microbiologists, social scientists, epidemiologists and stakeholders, in the form of two international workshops and one local workshop. The general finding of the DRA was the distinct lack of knowledge regarding a link between the presence of pathogens and diseases manifestation in sea turtles. A higher rate of disease in immunocompromised individuals was repeatedly reported and a possible link between immunosuppression and environmental contaminants as a result of anthropogenic influences was suggested. Society based conservation initiatives and as a result the cultural and social aspect of interacting with sea turtles appeared to need more attention and research. A risk management workshop was carried out to acquire the insights of local policy makers about management options for the risks relevant to Queensland and the options were evaluated considering their feasibility and effectiveness. The sea turtle DRA presented here, is a structured guide for future risk assessments to be used in specific scenarios such as translocation and head-starting programs

Disease risk analysis in sea turtles: a baseline study to inform conservation efforts

The impact of a range of different threats has resulted in the listing of six out of seven sea turtle species on the IUCN Red List of endangered species. Disease risk analysis (DRA) tools are designed to provide objective, repeatable and documented assessment of the disease risks for a population and measures to reduce these risks through management options. To the best of our knowledge, DRAs have not previously been published for sea turtles, although disease is reported to contribute to sea turtle population decline. Here, a comprehensive list of health hazards is provided for all seven species of sea turtles. The possible risk these hazards pose to the health of sea turtles were assessed and “One Health” aspects of interacting with sea turtles were also investigated. The risk assessment was undertaken in collaboration with more than 30 experts in the field including veterinarians, microbiologists, social scientists, epidemiologists and stakeholders, in the form of two international workshops and one local workshop. The general finding of the DRA was the distinct lack of knowledge regarding a link between the presence of pathogens and diseases manifestation in sea turtles. A higher rate of disease in immunocompromised individuals was repeatedly reported and a possible link between immunosuppression and environmental contaminants as a result of anthropogenic influences was suggested. Society based conservation initiatives and as a result the cultural and social aspect of interacting with sea turtles appeared to need more attention and research. A risk management workshop was carried out to acquire the insights of local policy makers about management options for the risks relevant to Queensland and the options were evaluated considering their feasibility and effectiveness. The sea turtle DRA presented here, is a structured guide for future risk assessments to be used in specific scenarios such as translocation and head-starting programs

Bacteriophage of Burkholderia pseudomallei; friend or foe?

Lysogenic bacteriophage carrying virulence determinants have been demonstrated to be responsible for the pathogenicity of many bacteria. Bacteriophage, or components of bacteriophage, have also been successfully used in the treatment of bacterial infections. Burkholderia pseudomallei is the causative agent of melioidosis and has been shown to carry bacteriophage. The role of bacteriophage in virulence of B. pseudomallei isolates has not yet been determined, nor have bacteriophage been examined for their potential in treatment of melioidosis. A screen for identification of bacterial isolates of interest was developed and 50 isolates were examined. Thirty-one selected isolates were then examined for bacteriophage using techniques including; transmission electron microscopy (T.E.M), mitomycin C assay, UV assay, plaque assay and restriction digestion assay. A combination of mitomycin C assay and either plaque assay or restriction digestion assay were determined to be 96.77% accurate for testing for bacteriophage in B. pseudomallei isolates. Five techniques for the concentration of bacteriophage (commercial Qiagen kit, magnesium hydroxide precipitation, PEG precipitation, zinc chloride precipitation, ultracentrifugation) were examined and ultracentrifugation determined to be the best. Two methods of DNA extraction (commercial nucleobond AX kit, phenol chloroform extraction) were compared and a phenol chloroform extraction was modified for use. A bacteriophage amplification system involving inoculation of bacteriophage into a broth of host B. pseudomallei, followed by lysis, was developed and optimised for production of lysogenic bacteriophage of B. pseudomallei. Addition of a 1:1 dose of bacteriophage to bacteria at an O.D.600nm of 0.1 in 10-100ml of broth resulted in the production of 1x1011plaque forming units (pfu)/ml of media upon lysis at 7.5 hours post-inoculation. Lysogenic bacteriophage extracted from highly virulent B. pseudomallei isolate NCTC 13178 was given the name BupsM1 and was characterised as being from the family Myoviridae with a genome 55.1kb long. This bacteriophage was then used for infection assays and molecular analysis to determine whether it played a role in virulence. Endolysin of this bacteriophage was also extracted to determine its potential for use in therapy. Four B. pseudomallei isolates tested negative for the presence of bacteriophage (#13, #69, #83, E4) and one isolate of particular interest (NAFC), were infected with BupsM1. Bacteriophage infection was found to alter colonial morphology on Ashdown agar. Infection assays in a BALB/c mouse model were carried out and no clear relationship between addition of bacteriophage BupsM1 and virulence was found. One experiment with NAFC resulted in greatly increased virulence, but this could not be repeated. All other experiments where infection with bacteriophage was successful resulted in minor upregulation or downregulation of virulence. Examination of plaque production of infected and control isolates indicated that prophage stability may play a role in survival of B. pseudomallei as addition of bacteriophage from NCTC13178 restored lysogenic stability to NAFC in several cases. Of the expected 55.1kb genome size from BupsM1, 51.3kb was sequenced with 40.9kb of this confirmed as bacteriophage. The open reading frames were determined using ORF finder and direct analysis. These open reading frames were analysed by BLASTx for putative function and several potential virulence genes were identified, as were structural, replication and lysogeny genes. Possible virulence genes include putative anaerobic dehydrogenase and oxidoreductase genes. Putative structural genes included the terminase large subunit, portal protein, head morphogenesis, tail assembly and tail fibre genes. Putative replication and lysogeny genes included transposases, insertion elements and integrase, an RNA polymerase sigma subunit, DNA cytosine methylase, Holliday junction resolvase, repressor protein, and a weak match to cro, the gene responsible for triggering lysis. Two genes of interest, the endolysin gene and a possible ADP-ribosyltransferase gene (a gene often involved in virulence) were not identified by BLASTx analysis. Techniques designed to identify genes with limited amino acid homology across species, such as identification of conserved amino acid pattern, chemo-physical comparison and phylogenetic tree analysis including bootstrap scoring, were then used to identify several open reading frames which were possible matches to these previously unidentified genes. The endolysin of BupsM1 was extracted under nine combinations of conditions from literature , using a natural host system (B. pseudomallei #4). EDTA was found to aid lysis, while chloroform was found to have no effect. Extracts were concentrated using Centricons™ and both neat and concentrated extracts were tested for their ability to lyse both killed and live B. pseudomallei #4 in broth and plate format. Neither the extracted endolysin nor its concentrate was found to lyse any of the B. pseudomallei in a form not attributable to live bacteriophage. Hence endolysin was determined not to function “from without” against B. pseudomallei. As such, this possibility for treatment of B. pseudomallei was eliminated

Isolation and examination of a bacteriophage of a pathogenic strain of Burkholderia pseudomallei

Information regarding the genomes of bacteriophage of Burkholderia pseudomallei is still limited with few genomes sequenced. Other bacterial species have been proved to have lysogenic bacteriophage that play a clear role in virulence, but this is not the case with B. pseudomallei (Summer et. al., 2007) and further investigation is warranted. In this work, a bacteriophage was isolated from a highly virulent strain of B. pseudomallei The bacteriophage caused alterations in colonial morphology of other B. pseudomallei isolates, but did not clear changes in virulence in a BALB/c mouse model. Examination of the majority of the phage genome was then carried out

A synthesis of the divalent cation requirements for efficient adsorption of bacteriophage onto bacterial cells

As the first step of bacteriophage infection, understanding the mechanics of adsorption of phages is vital for successful therapeutic phage treatments. It is also important for efficient scale-up of phages for industrial applications. It was understood early on that bacteriophage required divalent cations for successful adsorption. The mechanics of this process however are still vague; for some phage it may just be to negate negative electrostatic forces, for others it may also be specific for enzymatic and/or conformational changes. While many phages require divalent cations, the specificity of the ions and the concentrations differs between them. The adsorption efficiency of Ca2+ was superior in most studies to Mg2+ and other cations but the exact mechanisms are unresolved. The efficiency of Ca2+ might in general, explain why it is relatively easy to isolate phages from marine bacteria grown on marine salt based medium with high Ca2+/Mg2+ content. Too low a concentration of cations is insufficient for adsorption, and too high a concentration blocks adsorption. Of the monovalent cations, only H+ (pH) has been recorded to have a marked influence on adsorption. Phages become inactive and tend to aggregate at low pH and we hypothesise this is due to competitive exclusion interactions with Ca2+ or other bivalent cations. Whilst data are limited, the divalent cation optimum concentrations range between 0.01 and 10mM. Some phages like T4 phage may utilize organic substances such as L-tryptophan as a co-factor

Comparison of molecular detection PCR methods for chequa iflavirus in freshwater crayfish, Cherax quadricarinatus

Chequa iflavirus (+ve sense ssRNA virus) infects redclaw crayfish (Cherax quadricarinatus) and it may cause mortality reaching 20-40% after about three weeks following stress. The sequence of the RNA-dependent RNA polymerase at nucleotide position 8383-9873 was used for developing and comparing PCR-based detection protocols. The reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) was specific against nine Picornavirales and crustacean viruses and its' measurement of uncertainty (0.07-1.37) was similar to PCRs for other crustacean viruses. In vitro, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) read at 60 min had poor repeatability for a linearized plasmid with an iflavirus insert when compared with RT-PCR visualised on an electrophoretic gel and RT-qPCR; both sensitive to 10(2) copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88 x 10(4) for RT-PCR and 4.60 x 10(1) for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14 x 10(6). For RT-qPCR, the amplification plots, melt curves and the C-T values indicated that the C-T above 34.0 is a potential negative result but examination of the melt curve is necessary for an accurate interpretation. A suggested program of testing for crayfish farmers would consist of non-destructive bleeding, labelling of crayfish and screening with RT-qPCR. Only those crayfish nominally negative (below detectable limits) would be used for broodstock or selective breeding

Discovery of a novel Picornavirales, Chequa iflavirus, from stressed redclaw crayfish (Cherax quadricarinatus) from farms in northern Queensland, Australia

In 2014, northern Queensland crayfish from farms affected by particularly transportation and translocation stress, started to die with mortality reaching 20–40% after about three weeks and then mortalities subsided. Crayfish from 1 farm had 65% mortalities within 11 weeks. With histological examination of broodstock and juveniles, the muscle fibres were fractured with haemocytic infiltration reminiscent of viral infection or vitamin E/selenium deficiencies. Sequence dependent and independent PCRs failed to identify a viral aetiology. However, the whole transcriptomes of a case crayfish and an unaffected crayfish from a different population were assembled producing over 500,000 contigs. The complete sequence of a positive sense, single stranded RNA virus (+ve ssRNA virus; 9933 bp) and the large and medium segments of a bunya-like virus were detected. Transcript back-mapping and newly developed PCRs indicated that the viruses were in the case crayfish but not the control crayfish. The +ve ssRNA virus is clearly in the order Picornavirales, marginally in the genus Iflavirus in a clade of Chinese and Northern American terrestrial arthropod viruses. The internal Picornavirales motifs; RNA-dependent RNA polymerase, helicase (P-loop) and 2 viral capsids genes were easily identified. This is the first iflavirus identified from crustacea and is named Chequa iflavirus. Whether these viruses are responsible for the stress-related mortalities is unproven but the Chequa virus’ role seems limited as it appears it has been present in crayfish from at least the early 1990s; unless low-grade, chronic mortalities have been largely unnoticed

Assessment of a cricket, Acheta domesticus, bioassay for Chequa Iflavirus and bunya-like virus from redclaw crayfish Cherax quadricarinatus

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20–40% after about three weeks after a stress event. To complete River’s postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows >85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled as a bioassay animal. There was no significant difference (P > 0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30 days. The search for an alternative bioassay host will have to be widened

A synthesis of the divalent cation requirements for efficient adsorption of bacteriophage onto bacterial cells

As the first step of bacteriophage infection, understanding the mechanics of adsorption of phages is vital for successful therapeutic phage treatments. It is also important for efficient scale-up of phages for industrial applications. It was understood early on that bacteriophage required divalent cations for successful adsorption. The mechanics of this process however are still vague; for some phage it may just be to negate negative electrostatic forces, for others it may also be specific for enzymatic and/or conformational changes. While many phages require divalent cations, the specificity of the ions and the concentrations differs between them. The adsorption efficiency of Ca2+ was superior in most studies to Mg2+ and other cations but the exact mechanisms are unresolved. The efficiency of Ca2+ might in general, explain why it is relatively easy to isolate phages from marine bacteria grown on marine salt based medium with high Ca2+/Mg2+ content. Too low a concentration of cations is insufficient for adsorption, and too high a concentration blocks adsorption. Of the monovalent cations, only H+ (pH) has been recorded to have a marked influence on adsorption. Phages become inactive and tend to aggregate at low pH and we hypothesise this is due to competitive exclusion interactions with Ca2+ or other bivalent cations. Whilst data are limited, the divalent cation optimum concentrations range between 0.01 and 10mM. Some phages like T4 phage may utilize organic substances such as L-tryptophan as a co-factor