Confirmation that candidatus Coxiella cheraxi from redclaw crayfish (Cherax quadricarinatus) is a close relative of Coxiella burnetii, the agent of Q-fever.

A Coxiella sp. closely related to the agent of Q-fever, Coxiella burnetii, has been associated with mortalities in redclaw crayfish, (Cherax quadricarinatus), in farms and experimental facilities for three decades. Limited sequence data including 16S rRNA have placed the rickettsial species as a new species, candidatus C. cheraxi closely related to C. burnetii. MinION sequencing was conducted on the last remaining sample from an outbreak of disease, TO-98. The accuracy of base pair reads was mostly 99·9% (error rate 1 in 1000) or better. After filtering for reads of co-isolated Citrobacter freundii, 2629 sequences remained with the longest being 12 585 base pairs (bp). The longest 21 sequences are presented with their single best hit statistics when examined by NCBI blastn (nucleotides) and the nucleotides translated into proteins NCBI blastx. All sequences hit with either C. burnetii (29/42, 69%) or Coxiella (10/42, 24%) or rickettsia (3/42, 7%) with an error rate of less than 1 in 1 million for either bp or amino acids. Sequencing in this report confirms candidatus C. cheraxi is a new species very closely related to C. burnetii. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports on the use of newer technologies on archival samples and provides significantly more data on the currently limited genome data of candidatus Coxiella cheraxi, one of the few species isolated in the genus Coxiella. Candidatus Coxiella cheraxi causes death in redclaw crayfish and has been reported as being closely related to C. burnettii, the agent of Q-fever, based on 16S rRNA sequencing. This work provides confirmation for this claim.

The direct and indirect effects of kisspeptin-54 on granulosa lutein cell function.

STUDY QUESTION: What are the in vivo and in vitro actions of kisspeptin-54 on the expression of genes involved in ovarian reproductive function, steroidogenesis and ovarian hyperstimulation syndrome (OHSS) in granulosa lutein (GL) cells when compared with traditional triggers of oocyte maturation? SUMMARY ANSWER: The use of kisspeptin-54 as an oocyte maturation trigger augmented expression of genes involved in ovarian steroidogenesis in human GL cells including, FSH receptor (FSHR), LH/hCG receptor (LHCGR), steroid acute regulatory protein (STAR), aromatase, estrogen receptors alpha and beta (ESR1, ESR2), 3-beta-hydroxysteroid dehydrogenase type 2 (3BHSD2) and inhibin A (INHBA), when compared to traditional maturation triggers, but did not alter markers of OHSS. WHAT IS KNOWN ALREADY: hCG is the most widely used trigger of oocyte maturation, but is associated with an increased risk of OHSS. The use of GnRH agonists to trigger oocyte maturation is a safer alternative to hCG. More recently, kisspeptin-54 has emerged as a novel therapeutic option that safely triggers oocyte maturation even in women at high risk of OHSS. Kisspeptin indirectly stimulates gonadotropin secretion by acting on hypothalamic GnRH neurons. Kisspeptin and its receptor are also expressed in the human ovary, but there is limited data on the direct action of kisspeptin on the ovary. STUDY DESIGN SIZE, DURATION: Forty-eight women undergoing IVF treatment for infertility consented to kisspeptin-54 triggering and/or granulosa cell collection and were included in the study. Twelve women received hCG, 12 received GnRH agonist and 24 received kisspeptin-54 to trigger oocyte maturation. In the kisspeptin-54 group, 12 received one injection of kisseptin-54 (9.6 nmol/kg) and 12 received two injections of kisspeptin-54 at a 10 h interval (9.6 nmol/kg × 2). PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid was aspirated and pooled from follicles during the retrieval of oocytes for IVF/ICSI. GL cells were isolated and either RNA extracted immediately or cultured in vitro ± kisspeptin or hCG. MAIN RESULTS AND THE ROLE OF CHANCE: GL cells from women who had received kisspeptin-54 had a 14-fold and 8-fold higher gene expression of FSHR and a 2-fold (ns) and 2.5-fold (P < 0.05) higher expression of LHCGR than GL cells from women who had received hCG or GnRH agonist, respectively. CYP19A1 expression was 3.6-fold (P < 0.05) and 4.5-fold (P < 0.05) higher, STAR expression was 3.4-fold (P < 0.01) and 1.8-fold (P < 0.05) higher, HSD3B2 expression was 7.5- (P < 0.01) and 2.5-fold higher (P < 0.05), INHBA was 2.5-fold (P < 0.01) and 2.5-fold (P < 0.01) higher in GL cells from women who had received kisspeptin-54 than hCG or GnRHa, respectively. ESR1 (P < 0.05) and ESR2 (P < 0.05) both showed 3-fold higher expression in cells from kisspeptin treated than GnRHa treated women. Markers of vascular permeability and oocyte growth factors were unchanged (VEGFA, SERPINF1, CDH5, amphiregulin, epiregulin). Gene expression of kisspeptin receptor was unchanged. Whereas treating GL cells in vitro with hCG induced steroidogenic gene expression, kisspeptin-54 had no significant direct effects on either OHSS genes or steroidogenic genes. LIMITATIONS REASONS FOR CAUTION: Most women in the study had PCOS, which may limit applicability to other patient groups. For the analysis of the in vitro effects of kisspeptin-54, it is important to note that GL cells had already been exposed in vivo to an alternate maturation trigger. WIDER IMPLICATIONS OF THE FINDINGS: The profile of serum gonadotropins seen with kisspeptin administration compared to other triggers more closely resemble that of the natural cycle as compared with hCG. Thus, kisspeptin could potentially permit an ovarian environment augmented for steroidogenesis, in particular progesterone synthesis, which is required for embryo implantation. STUDY FUNDING/COMPETING INTEREST(S): Dr Owens is supported by an Imperial College London PhD Scholarship. Dr Abbara is supported by an National Institute of Health Research Academic Clinical Lectureship. The authors do not have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01667406.

Mosquito cells (C6/36) fail to support the complete replication of Penaeus merguiensis hepandensovirus.

Mosquito cell lines (C6/36) were reported in the literature to support the propagation of Penaeus monodon hepandensovirus (PmoHDV). We aim to evaluate the susceptibility and viral propagation of P. merguiensis hepandensovirus (PmeHDV) which is ∼22% different to PmoHDV in Aedes albopictus cell line (C6/36). Cellular changes in the infected cell culture were detected. Vacuole formation was seen in both infected and uninfected cell cultures. The average number of disrupted cellular membranes in the infected cells (presumptive dead cells) was significantly higher than that of uninfected cells at passage two (F=9.749, d.f. 1, 22, p<0.05). Using a proliferation assay, light absorption of infected cells peaked at 2weeks post-infection (O.D.=0.27) but was significantly lower than that of the uninfected groups (O.D.=0.37) (F=6.879, d.f. 1, 94, p<0.05) suggesting hindered cell growth. PCR of the serial passages of the infected cell cultures indicated weak positive results for PmeHDV infection and TaqMan quantitative PCR confirmed that the average number of viral copies declined from 3.8×105 to 5.69×102 copies per µL and the mean of cycle times increased from 19.26 to 27.63. These results are interpreted to mean C6/36 allows the initial stage of PmeHDV replication, but the virus was incapable of using C6/36 for patent replication of its' virions.

Serological survey of Australian native reptiles for exposure to ranavirus.

Ranaviruses have been isolated from many ectothermic vertebrates, and serological surveys of both amphibians and reptiles have shown the presence of ranaviral antibodies in a proportion of these populations. An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum antibodies against ranavirus in Australian reptiles. The ELISA was validated with serum from challenge trials with Bohle iridovirus (BIV) in 6 reptilian species. A preliminary sero-survey of northern Queensland riparian reptile fauna (saw-shelled turtles Myuchelys latisternum, Krefft's river turtles Emydura macquarii krefftii, freshwater crocodiles Crocodylus johnstoni, as well as the snakes Boiga irregularis, Dendrelaphis punctulatus, Tropidonophis mairii, Morelia spilota, Liasis childreni and L. fuscus) revealed evidence of past exposure to Bohle iridoviral antigens in part of the population at several locations sampled. Furthermore, in Krefft's river turtles and freshwater crocodiles, a statistically significant trend was apparent for larger reptiles to be more likely to have BIV-reactive sera than smaller individuals. The use of adult tortoise populations as sentinels can assist in monitoring the presence of BIV in northern Australian freshwater streams, and thereby the potential dangers to native fauna from this agent.

Environmental Exposures-The Missing Link in Immune Responses After Transplantation.

In transplantation, immunosuppression has been directed at controlling acute responses, but treatment of chronic rejection has been ineffective. It is possible that factors that have previously been unaccounted for, such as exposure to inhaled pollution, ultraviolet light, or loss of the normal equilibrium between the gut immune system and the outside environment may be responsible for shifting immune responses to an effector/inflammatory phenotype, which leads to loss of self-tolerance and graft acceptance, and a shift towards autoimmunity and chronic rejection. Cells of the immune system are in a constant balance of effector response, regulation, and quiescence. Endogenous and exogenous signals can shift this balance through the aryl hydrocarbon receptor, which serves as a thermostat to modulate the response one way or the other, both at mucosal surfaces of interface organs to the outside environment, and in the internal milieu. Better understanding of this balance will identify a target for maintenance of self-tolerance and continued graft acceptance in patients who have achieved a "steady state" after transplantation.

Development of the Alcohol Quality of Life Scale (AQoLS): a new patient-reported outcome measure to assess health-related quality of life in alcohol use disorder.

PURPOSE: The development of patient-centred approaches and of reduction in consumption strategies in alcohol use disorder requires giving a larger place to qualitative assessments that are closer to patients' concerns and more clinically relevant than drinking outcomes and generic health-related quality of life instrument. Our purpose was to develop from patients input the Alcohol Quality of Life Scale (AQoLS), a disease-specific measure for alcohol use disorder (AUD). METHODS: Concept elicitation focus groups were conducted with AUD patients in the UK and France. Thematic analysis was used to identify key areas of impact of AUD, and draft items were developed to capture these issues. The draft items underwent expert review to ensure clinical and cross-cultural applicability. Two iterative rounds of cognitive debriefing interviews were conducted with AUD patients in both countries, to assess face and content validity. RESULTS: From focus groups conducted with 38 AUD patients, seven areas of impact emerged, forming the basis for the AQoLS: relationships, activities, looking after self, emotional impact, control, living conditions, and sleep. An initial pool of 90 items was reduced to 50 following the review process. In cognitive interviews, the measure took less than 10 min to complete, and patients reported that items were relevant to their experience. Following Round 1 interviews (n = 16), 14 items were removed because patients felt they were unclear or uneasy to respond to, 2 were combined, 7 were revised, and 4 new items were added. The recall period of 2 weeks was changed to 4 weeks, based on patient comments. Following Round 2 interviews (n = 15), 5 items were removed and 3 were modified to produce the 34-item AQoLS. CONCLUSION: As the only de novo measure of health-related quality of life developed specifically for AUD, the AQoLS offers the potential of increased sensitivity to show the effectiveness of therapeutic interventions from the patient's perspective.

Pathogenicity in six Australian reptile species following experimental inoculation with Bohle iridovirus.

Ranaviruses are able to infect multiple species of fish, amphibian and reptile, and some strains are capable of interclass transmission. These numerous potential carriers and reservoir species compound efforts to control and contain infections in cultured and wild populations, and a comprehensive knowledge of susceptible species and life stage is necessary to inform such processes. Here we report on the challenge of 6 water-associated reptiles with Bohle iridovirus (BIV) to investigate its potential pathogenicity in common native reptiles of the aquatic and riparian fauna of northern Queensland, Australia. Adult tortoises Elseya latisternum and Emydura krefftii, snakes Boiga irregularis, Dendrelaphis punctulatus and Amphiesma mairii, and yearling crocodiles Crocodylus johnstoni were exposed via intracoelomic inoculation or co-habitation with infected con-specifics, but none were adversely affected by the challenge conditions applied here. Bohle iridovirus was found to be extremely virulent in hatchling tortoises E. latisternum and E. krefftii via intracoelomic challenge, as demonstrated by distinct lesions in multiple organs associated with specific immunohistochemistry staining and a lethal outcome (10/17) of the challenge. Virus was re-isolated from 2/5 E. latisternum, 4/12 E. krefftii and 1/3 brown tree snakes B. irregularis. Focal necrosis, haemorrhage and infiltration of granulocytes were frequently observed histologically in the pancreas, liver and sub-mucosa of the intestine of challenged tortoise hatchlings. Immunohistochemistry demonstrated the presence of ranavirus antigens in the necrotic lesions and in individual cells of the vascular endothelium, the connective tissue and in granulocytes associated with necrosis or present along serosal surfaces. The outcome of this study confirms hatchling tortoises are susceptible to BIV, thereby adding Australian reptiles to the host range of ranaviruses. Additionally, given that BIV was originally isolated from an amphibian, our study provides additional evidence that interclass transmission of ranavirus may occur in the wild.

Induction and characterization of lysogenic bacteriophages from Streptococcus iniae.

AIMS: To investigate the presence of prophage in Streptococcus iniae, a highly problematic fish pathogen. METHODS AND RESULTS: Cross-spotting assays and mitomycin C inductions were conducted to screen for prophage in 48 Strep. iniae isolates. Bacteriophages were characterized by plaque assays, transmission electron microscopy and DNA restriction enzyme digestion. Plaque assays confirmed prophages in 14·6% of isolates. Phages vB_SinS-44, vB_SinS-45, vB_SinS-46 and vB_SinS-48 lysed 78·5% of Strep. iniae isolates and displayed distinctive host ranges. Microscopy revealed virions exhibiting long, non-contractile tails and isometric heads consistent with phages from the family Siphoviridae. Restriction digests revealed genome sizes ranging from 27·5 to 66·3 kbp, with distinct cutting patterns that indicate the presence of related prophages in bacteria isolated from different geographic regions. CONCLUSIONS: The rate of prophage carriage found is comparably low and induction rates varied between phages. The four characterized Siphoviridae phages have broad host ranges within the Strep. iniae isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description and characterization of lysogenic phages from Strep. iniae. These phages are candidates for research and diagnosis of the bacterium and their identification should accelerate the discovery of lytic phages to be trialled against Strep. iniae infections in fish.

Non-permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus.

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi-square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10(5)  cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10(5)  cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10(4) and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.

Bacteriophage adenine methyltransferase: a life cycle regulator? Modelled using Vibrio harveyi myovirus like.

The adenine methyltransferase (DAM) gene methylates GATC sequences that have been demonstrated in various bacteria to be a powerful gene regulator functioning as an epigenetic switch, particularly with virulence gene regulation. However, overproduction of DAM can lead to mutations, giving rise to variability that may be important for adaptation to environmental change. While most bacterial hosts carry a DAM gene, not all bacteriophage carry this gene. Currently, there is no literature regarding the role DAM plays in life cycle regulation of bacteriophage. Vibrio campbellii strain 642 carries the bacteriophage Vibrio harveyi myovirus like (VHML) that has been proven to increase virulence. The complete genome sequence of VHML bacteriophage revealed a putative adenine methyltransferase gene. Using VHML, a new model of phage life cycle regulation, where DAM plays a central role between the lysogenic and lytic states, will be hypothesized. In short, DAM methylates the rha antirepressor gene and once methylation is removed, homologous CI repressor protein becomes repressed and non-functional leading to the switching to the lytic cycle. Greater understanding of life cycle regulation at the genetic level can, in the future, lead to the genesis of chimeric bacteriophage with greater control over their life cycle for their safe use as probiotics within the aquaculture industry.

Inputs and losses by surface runoff and subsurface leaching for pastures managed by continuous or rotational stocking.

Pasture management practices can affect forage quality and production, animal health and production, and surface and groundwater quality. In a 5-yr study conducted at the North Appalachian Experimental Watershed near Coshocton, Ohio, we compared the effects of two contrasting grazing methods on surface and subsurface water quantity and quality. Four pastures, each including a small, instrumented watershed (0.51-1.09 ha) for surface runoff measurements and a developed spring for subsurface flow collection, received 112 kg N ha(-1) yr(-1) and were grazed at similar stocking rates (1.8-1.9 cows ha(-1)). Two pastures were continuously stocked; two were subdivided so that they were grazed with frequent rotational stocking (5-6 times weekly). In the preceding 5 yr, these pastures received 112 kg N ha(-1) yr(-1) after several years of 0 N fertilizer and were grazed with weekly rotational stocking. Surface runoff losses of N were minimal. During these two periods, some years had precipitation up to 50% greater than the long-term average, which increased subsurface flow and NO(3)-N transport. Average annual NO(3)-N transported in subsurface flow from the four watersheds during the two 5-yr periods ranged from 11.3 to 22.7 kg N ha(-1), which was similar to or less than the mineral-N received in precipitation. Flow and transport variations were greater among seasons than among watersheds. Flow-weighted seasonal NO(3)-N concentrations in subsurface flow did not exceed 7 mg L(-1). Variations in NO(3)-N leached from pastures were primarily due to variable precipitation rather than the effects of continuous, weekly rotational, or frequent rotational stocking practices. This suggests that there was no difference among these grazing practices in terms of NO(3)-N leaching.

The impact of travel distance on the decision to attend for screening for gestational diabetes mellitus.

This paper estimates the impact of travel distance on the decision to attend for screening for gestational diabetes mellitus (GDM), controlling for a range of personal, clinical and lifestyle characteristics. The results suggest that women who live further away from a screening site are less likely to attend for screening. In particular, the probability of attending for screening is reduced by 1.8% [95% CI: 1.2% to 2.4%] for every additional 10 kms of travel. This is consistent wth previous research that shows geographic inequalities in access to GDM screening in Ireland. We also find that older women, those with a family history of diabetes, and those who are obese are more likely to accept the screening offer, suggesting that certain higher-risk groups may be either self-selecting into the screening programme or are being targeted by health care professionals through specific initiatives.

Is there a socioeconomic gradient in the prevalence of gestational diabetes mellitus?

Previous studies have shown an association between Type 2 diabetes and lower socioeconomic status. This link is less clear in those with gestational diabetes mellitus (GDM). We test for a socioeconomic gradient in the prevalence of GDM by analysing data on 9,842 pregnant women who were offered testing for GDM in the Atlantic Diabetes in Pregnancy universal screening programme. A bivariate probit model relating GDM prevalence to socioeconomic status was estimated, controlling for variation in screening uptake rates across socioeconomic groups. The estimated increased prevalence of GDM is 8.6% [95% CI 2.7%-12.0%] for women in the lowest socioeconomic group when compared to the highest, suggesting a strong socioeconomic gradient in the prevalence of GDM. This gradient is found to be driven by differences in personal, clinical and lifestyle factors across socioeconomic groups.

Changing clinical practice can improve clinical outcomes for women with pre-gestational diabetes mellitus.

Prospective evaluation of pregnancy outcomes in women with pre-gestational diabetes over 6 years. The ATLANTIC Diabetes in Pregnancy group represents 5 antenatal centres along the Irish Atlantic seaboard, providing care for women with diabetes throughout pregnancy. In 2007 the group published a report that recognised that women were poorly prepared for pregnancy and that outcomes were sub-optimal. A change in practice occurred, offering women specialist-led, evidence-based care, both pre-pregnancy and combined antenatal/diabetes clinics during pregnancy. We now compare outcomes from 2005-2007 with 2008-2010. There was an increase in the numbers attending pre-conception care. Glycemic control before and throughout pregnancy improved. There was an overall increase in live births and decrease in perinata mortality rate. There was a decrease in large-for-gestational-age babies in mothers with Type 1 Diabetes. Elective Caesarean section rates increased while emergency section rates decreased. More women had Type 2 diabetes over time and these women were more likely to be obese. Changing the process of clinical care delivery can improve outcomes in for women with pre-gestational diabetes.

Psychological stress associated with diabetes during pregnancy: a pilot study.

Gestational Diabetes Mellitus (GDM) affects approximately 12% of women. The impact of a diagnosis of GDM may lead to increased stress in pregnancy due to the demands of adherence to a treatment regimen and maternal concern about adverse outcomes for the mother and baby. We examined the psychosocial profile of 25 women with gestational diabetes mellitus (GDM) and compared them to 25 non-diabetic pregnant women. Measures administered included the Pregnancy Experiences Scale (PES), the Depression, Anxiety Stress Scale (DASS), the Problem Areas in Diabetes Scale (PAID-5) and the Perceived Social Support Scale (PSSS). The GDM group reported a significantly greater ratio of pregnancy 'hassles' to pregnancy 'uplifts'. The GDM group also had a significantly higher Depression score and were twice as likely to score above the cut-off for possible depression. Elevated levels of diabetes-related distress were found in 40% of women with GDM. In addition, the GDM group reported less social support from outside the family. Our preliminary study indicates that the experience of GDM appears to be associated with increased psychological distress in comparison to the experience of non-diabetic pregnant women. This may indicate the need for psychological screening in GDM and the provision of psychological support in some cases.

Trimester-specific reference intervals for IFCC standardised haemoglobin A(1c): new criterion to diagnose gestational diabetes mellitus (GDM)?

We established trimester-specific reference intervals for IFCC standardised HbA(1c) in 311 non-diabetic Caucasian pregnant women (n = 246) and non-pregnant women (n = 65). A selective screening strategy based on risk factors for gestational diabetes was employed. Pregnancy trimester was defined as trimester 1 (T1, n = 40) up to 12 weeks + 6 days, trimester 2 (T2, n = 106) 13 to 27 weeks + 6 days, trimester 3 (T3, n = 100) > 28 weeks to delivery. The normal HbA(1c) reference interval for Caucasian non-pregnant women was 29-37 mmol/mol (DCCT: 4.8-5.5%), T1: 24-36 mmol/mol (DCCT: 4.3-5.4%), T2: 25-35 mmol/mol (DCCT: 4.4-5.4%), and T3: 28-39 mmol/mol (DCCT: 4.7-5.7%). HbA(1c) was significantly decreased in trimesters 1 (P < 0.01) and 2 (P < 0.001) compared to non-pregnant women. Retrospective application of selective screening to Caucasian women of the Atlantic DIP cohort determined that 5,208 met the criteria. 945 of those women (18.1%) were diagnosed with Gestational Diabetes Mellitus (GDM) using the International Association of Diabetes and Pregnancy Study Groups (IADPSG) glucose concentration thresholds. HbA(1c) measurement within 2 weeks of the diagnostic Oral Glucose Tolerance Test (OGTT) was available in 622 of 945 (66%). Applying the decision threshold for T2: HbA(1c) > 35 mmol/mol (DCCT > 5.4%) identified 287 of 622 (46%) of those with GDM. HbA(1c) measurement in T2 (13 to 27 weeks) should be included in the diagnostic armamentarium for GDM. This would reduce the need for diagnostic OGTT in a significant number of women.

Injection of Acanthaster planci with thiosulfate-citrate-bile-sucrose agar (TCBS). II. Histopathological changes.

We assessed histological changes in the tissues of the crown-of-thorns starfish Acanthaster planci (COTS) after injection of thiosulfate-citrate-bile-sucrose agar (TCBS) which was used as a disease inducer (potential outbreak control method), by conventional and scanning electron microscopy. Digestive glands were processed and stained with hematoxylin and eosin to describe the histological architecture of the intestinal epithelium. Subsequently comparison of healthy versus infected tissues and Gram stains were carried out to confirm bacterial occurrence on infected tissues, characterize the structural changes induced by bacterial communities in COTS tissues, and to determine if the histopathological changes of intestinal tissues were consistent with vibrio infection. TCBS injections induced marked epithelial desquamation, hypertrophy and hypersecretion of glandular cells, epithelial cell destruction, pyknosis, reduction of thickness and disorganization of connective tissue and associated nerve plexus, presence of bacterial colonies, irregular eosinophilic foci in glandular cells, brush border disruption, atrophy and detachment of intestinal microvilli and cell debris in the lumen. All these changes were attributed to a fulminating systemic dysbiosis and were consistent with vibrio infections.

Refined identification of Vibrio bacterial flora from Acanthasther planci based on biochemical profiling and analysis of housekeeping genes.

We used a polyphasic approach for precise identification of bacterial flora (Vibrionaceae) isolated from crown-of-thorns starfish (COTS) from Lizard Island (Great Barrier Reef, Australia) and Guam (U.S.A., Western Pacific Ocean). Previous 16S rRNA gene phylogenetic analysis was useful to allocate and identify isolates within the Photobacterium, Splendidus and Harveyi clades but failed in the identification of Vibrio harveyi-like isolates. Species of the V harveyi group have almost indistinguishable phenotypes and genotypes, and thus, identification by standard biochemical tests and 16S rRNA gene analysis is commonly inaccurate. Biochemical profiling and sequence analysis of additional topA and mreB housekeeping genes were carried out for definitive identification of 19 bacterial isolates recovered from sick and wild COTS. For 8 isolates, biochemical profiles and topA and mreB gene sequence alignments with the closest relatives (GenBank) confirmed previous 16S rRNA-based identification: V. fortis and Photobacterium eurosenbergii species (from wild COTS), and V natriegens (from diseased COTS). Further phylogenetic analysis based on topA and mreB concatenated sequences served to identify the remaining 11 V harveyi-like isolates: V. owensii and V. rotiferianus (from wild COTS), and V. owensii, V. rotiferianus, and V. harveyi (from diseased COTS). This study further confirms the reliability of topA-mreB gene sequence analysis for identification of these close species, and it reveals a wider distribution range of the potentially pathogenic V. harveyi group.

Injection of Acanthaster planci with thiosulfate-citrate-bile-sucrose agar (TCBS). I. Disease induction.

This is the first report of the successful induction of a transmissible disease in the coral-eating crown-of-thorns starfish Acanthaster planci (COTS). Injection of thiosulfate-citrate-bile-sucrose agar (TCBS) culture medium into COTS induced a disease characterized by discoloured and necrotic skin, ulcerations, loss of body turgor, accumulation of colourless mucus on many spines especially at their tip, and loss of spines. Blisters on the dorsal integument broke through the skin surface and resulted in large, open sores that exposed the internal organs. Oedema and reddened digestive tissues and destruction of connective fibers were common. Moreover, healthy COTS in contact with these infected animals also displayed signs of disease and died within 24 h. TCBS induced 100% mortality in injected starfish. There was no introduction of new pathogens into the marine environment. TCBS promoted the growth of COTS' naturally occurring Vibrionales to high densities with subsequent symbiont imbalance followed by disease and death.

Gill-associated virus and its association with decreased production of Penaeus monodon in Australian prawn farms.

Gill-associated virus (GAV) was found to be associated with decreased prawn, Penaeus monodon, production when prawns from three farms (n = 45 ponds, 1800 prawns) were monitored for GAV over the production season using a graded RT-nPCR. The grading system used was a visualization of either the outer or inner nested PCR products. Prevalence and loading of GAV were associated with disease severity. Ponds with a higher initial prevalence and a larger increase in GAV load over the production period suffered disease outbreaks. Ponds with low initial prevalence of GAV but a larger increase in prevalence and large increase in load over the production period suffered chronic disease with no disease outbreak identified, yet low production. However, the ponds with moderate to low initial prevalence of GAV with a low increase in prevalence and load of GAV over the production period incurred no disease outbreak and comparatively high production. Ponds with GAV prevalence greater than 75% at 1 month post-stocking should be considered for termination as they have a high probability (95%) of having a disease outbreak. Emergency harvest when a disease outbreak occurs will significantly limit mortality losses.