Rapid Diagnostic Test to Detect and Discriminate Infectious Hematopoietic Necrosis Virus (IHNV) Genogroups U and M to Aid Management of Pacific Northwest Salmonid Populations.

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe, and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E, and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture, the widely accepted reference standard, as well as the previously validated universal N RT-rPCR test. The high diagnostic performance of the new U/M assay indicates the test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap.

Abalone Withering Syndrome Disease Dynamics: Infectious Dose and Temporal Stability in Seawater.

Withering syndrome (WS) is a chronic bacterial disease that affects numerous northeastern Pacific abalone Haliotis spp. The causative agent of WS is an obligate intracellular Rickettsiales-like bacterium (WS-RLO) that remains unculturable, thereby limiting our understanding of WS disease dynamics. The objectives of our study were to (1) determine the temporal stability of WS-RLO DNA outside of its abalone host in 14°C and 18°C seawater, (2) develop a standardized protocol for exposing abalones to known concentrations of WS-RLO DNA, and (3) calculate the dose of WS-RLO DNA required to generate 50% infection prevalence (ID50) in the highly cultured red abalone Haliotis rufescens. The WS-RLO stability trials were conducted in October 2016, February 2017, and June 2017. A quantitative PCR (qPCR) analysis was used to quantify bacterial DNA for 7 d in seawater collected at an abalone farm in southern California, where the pathogen is now endemic. For all trials and temperature treatments, WS-RLO DNA was unstable in seawater for longer than 2 d. To determine an ID50, groups of uninfected juvenile red abalone were subjected to 3-h bath exposures with four concentrations of WS-RLO at 0, 103 , 104 , and 105 DNA copies/mL. Abalone feces were tested biweekly for the presence of WS-RLO DNA, and abalone tissues were sampled 9 weeks postinfection for histological and qPCR analyses. The ID50 results indicated that our protocol was successful in generating WS-RLO infections; a pathogen dose of 2.3 × 103 DNA copies/mL was required to generate a 50% infection prevalence in red abalone tissue. These findings are critical components of disease dynamics that will help assess WS transmission risk within and among abalone populations and facilitate appropriate management and restoration strategies for both wild and cultured abalone species in WS-endemic areas.

Withering syndrome susceptibility of northeastern Pacific abalones: A complex relationship with phylogeny and thermal experience.

Population declines in wild and cultured abalones (Haliotis spp.) due to a bacterial disease called withering syndrome (WS) have been documented along the northeastern Pacific Ocean. However, observed differences in species susceptibility to the disease are not well understood. Here, we examined the susceptibility of three temperate abalone species, the cool water (4-14 °C) pinto or northern abalone (Haliotis kamtschatkana), the intermediate water (8-18 °C) red abalone (H. rufescens), and the warm water (12-23 °C) pink abalone (H. corrugata), to experimental WS infection at temperatures facilitating disease proliferation. Mortality data paired with histological and molecular detection of the WS pathogen confirmed that these abalone species exhibit different levels of susceptibility to infection and resistance to WS development ranging from high susceptibility and low resistance in pinto abalone to moderate/low susceptibility and resistance in red and pink abalones. The temperature associated with WS induced mortalities also varied among species: pinto abalone died at the lowest experimental temperature (17.32 ±â€¯0.09 °C), while red abalone died at an intermediate temperature (17.96 ±â€¯0.16 °C), and pink abalone required the highest temperature (18.84 ±â€¯0.16 °C). When data from the current and previous studies were examined, susceptibility to WS was inversely related to phylogenetic distance from white abalone (H. sorenseni), which had the highest susceptibility and lowest resistance of all abalone species tested prior to the current study. These results provide further evidence that an abalone's thermal optima and phylogenetic relationship can determine its susceptibility to WS; species with cool water evolutionary histories are most susceptible to WS and the most susceptible species appear to be closely related. Differences among the thermal ranges of abalone species have broad implications for WS disease dynamics and highlight the importance of understanding the mechanisms governing the abalone-WS relationship in order to properly manage declining abalone populations.

Validation of a quantitative PCR assay for detection and quantification of 'Candidatus Xenohaliotis californiensis'.

Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments.

Abalone withering syndrome: distribution, impacts, current diagnostic methods and new findings.

Withering syndrome (WS) is a fatal disease of abalone caused by a Rickettsiales-like organism (WS-RLO). The causative agent, 'Candidatus Xenohaliotis californiensis', occurs along the eastern Pacific margin of North America in California, USA, and Baja California, Mexico. However, as infected abalones have been transported to Chile, China, Taiwan, Iceland, Ireland, Israel, Spain, Thailand and Japan, the geographical range of the etiological agent is suspected to be broad, especially where California red abalones Haliotis rufescens are cultured or in areas where native species have been exposed to this species. Susceptibility varies among species, with up to 99% losses of black abalone H. cracherodii in laboratory and field studies in the USA to no losses among the small abalone H. diversicolor supertexta in Thailand. Some populations that have suffered catastrophic losses due to WS have developed resistance to the disease. In addition, a newly identified phage hyperparasite of the WS-RLO may reduce pathogenicity and dampen associated losses. Diagnosis of WS requires the identification of infection with the pathogen (WS-RLO detected via in situ hybridization or histology coupled with PCR and sequence analysis) accompanied by morphological changes that characterize this disease (e.g. pedal and digestive gland atrophy, and digestive gland metaplasia). A quantitative PCR assay was developed and may be useful in quantifying pathogen DNA. Confirmation of infection cannot be done by PCR analysis alone but can be used as a proxy for infection in areas where the agent is established and is recommended for inclusion in health examinations. Avoidance of WS is best accomplished by the establishment of a health history and multiple health examinations prior to movement of animals.

Reduced disease in black abalone following mass mortality: phage therapy and natural selection.

Black abalone, Haliotis cracherodii, populations along the NE Pacific ocean have declined due to the rickettsial disease withering syndrome (WS). Natural recovery on San Nicolas Island (SNI) of Southern California suggested the development of resistance in island populations. Experimental challenges in one treatment demonstrated that progeny of disease-selected black abalone from SNI survived better than did those from naïve black abalone from Carmel Point in mainland coastal central California. Unexpectedly, the presence of a newly observed bacteriophage infecting the WS rickettsia (WS-RLO) had strong effects on the survival of infected abalone. Specifically, presence of phage-infected RLO (RLOv) reduced the host response to infection, RLO infection loads, and associated mortality. These data suggest that the black abalone: WS-RLO relationship is evolving through dual host mechanisms of resistance to RLO infection in the digestive gland via tolerance to infection in the primary target tissue (the post-esophagus) coupled with reduced pathogenicity of the WS-RLO by phage infection, which effectively reduces the infection load in the primary target tissue by half. Sea surface temperature patterns off southern California, associated with a recent hiatus in global-scale ocean warming, do not appear to be a sufficient explanation for survival patterns in SNI black abalone. These data highlight the potential for natural recovery of abalone populations over time and that further understanding of mechanisms governing host-parasite relationships will better enable us to manage declining populations.

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

Putative phage hyperparasite in the rickettsial pathogen of abalone, "Candidatus Xenohaliotis californiensis".

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host-parasite interactions and associated microbial community structure. The marine rickettsial organism, "Candidatus Xenohaliotis californiensis" (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9-99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are "Candidatus Xenohaliotis californiensis," and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.