Rickettsia-like Organisms Research Articles

Mass mortality of Cornu aspersum in Italian snail farms: A histopathological survey

The production demand of edible snails in the Mediterranean area is very high and the attention to snail borne diseases is increasing. Following mass mortality events, we have analyzed 240 samples of Cornu aspersum collected from farms across Italy. Anatomopathological examination showed the presence of alterations of the gastro-intestinal apparatus and of the digestive gland, while histopathological examination revealed the presence of Rickettsia-like organisms (RLOs) in 70% (168/240) of cases and Giemsa positive amoebae in the remaining 30% (72/240) of cases. RLOs were localized mainly at the level of the DG, where regressive changes or nodular inflammation was observed. TEM examination of RLOs samples revealed the presence of many rod-shaped electron dense microorganisms. Amoebal infection occurred in the kidney, intestine, lung, the DG and were associated to regressive events or infiltrative/nodular and encapsulation like inflammation. To date it is still unclear if the pathogens detected could represent a risk for humans and animals, therefore further studies are needed to better elucidate this point.

Prevalence and intensity of a Rickettsiales-like organism in cultured pleasure oyster, Crassostrea corteziensis, from Nayarit, Mexico

Fastidious endosymbiotic Rickettsiales-like organisms (RLOs) have been observed in the digestive diverticula of the cultured pleasure oyster (Crassostrea corteziensis) from Nayarit, Mexico since 2007. In a few mollusk species, these bacteria have been associated with mortality events and production losses. The type of relationship between the RLOs and the pleasure oyster is largely unknown and further investigations are needed to determine if these bacteria warrant management concern in C. corteziensis. In this study, the morphological characteristics of the RLOs were studied by histology and SEM, and the taxonomic affiliations of the bacteria were evaluated by 16S rRNA amplicon sequencing. In addition, the prevalence and intensity of the RLOs was recorded from 2007 to 2017 by histology. The RLOs were observed inside circular basophilic cytoplasmic membrane bound vacuoles (MBVs) that had an average length and width of 15.70 ± 15.24 µm and 15.42 ± 14.95 µm respectively. Apart from cellular hypertrophy, no tissue alterations were observed in the areas adjacent to the RLOs. Individual bacteria within the MBVs were coccoid in shape with an average length of 0.65 ± 0.12 µm and an average width of 0.38 ± 0.09 µm. The bacterial microbiota of a selected number of samples (one sample without RLOs and two samples with RLOs) showed the presence of intracellular parasite OTUs corresponding to the families Rickettsiaceae and Anaplasmataceae, suggesting that the RLOs from the pleasure oyster is associated with the order Rickettsiales. A mean prevalence of 5 % was observed throughout the study period and the majority of the organisms (89 %) presented low intensity of Grade 1 (30–61 RLOs) of the MBVs. A higher prevalence of the RLOs was observed during warmer months. The lack of tissue alterations, the low prevalence and the low intensity of the MBVs suggest that the RLOs from C. corteziensis is a commensal endosymbiont that presents little risk for oyster production in Nayarit, México. However, regular monitoring is needed to detect if any variation in this relationship occurs, mainly in a scenario where extreme environmental fluctuations may occur.

New Zealand rickettsia-like organism and Tenacibaculum maritimum vaccine efficacy study.

A cohort of Chinook salmon juveniles was vaccinated, with an autogenous bivalent vaccine against New Zealand RLOs (NZ-RLO1) and Tenacibaculum maritimum. A proportion of the cohort was not vaccinated to act as controls. At smoltification, the fish were challenged with NZ-RLO1, NZ-RLO2, or T. maritimum. We found that challenge with T. maritimum by immersion in (7.5 × 105 cfu/mL of water) did not yield any pathology. Challenge with RLOs produced clinical signs that were more or less severe depending on the challenge route, dose or vaccination status. Survival was significantly higher for vaccinated fish within the groups challenged with NZ-RLO1 by intraperitoneal injection with a relative percent survival (RPS) of 48.84%. Survival was not significantly different between vaccinated and non-vaccinated fish for groups challenged with NZ-RLO2 by intraperitoneal injection or by NZ-RLO1 by immersion. Yet, anecdotally the clinical disease presentation (manifesting as haemorrhagic, ulcerative skin lesions) was more severe for the non-vaccinated fish. This study demonstrates that autogenous vaccine against NZ-RLO is protective against severe disease and death by NZ-RLO1 challenge which warrants implementation and further evaluation under field conditions. Yet, this study also highlights the importance of the route of administration and dose when evaluating pathogenicity and vaccine efficacy.

Diagnosis of Anaplasma marginale in cattle at the Iowa State University veterinary diagnostic laboratory 2003–2021

Anaplasma marginale is a blood-borne rickettsia-like organism that infects cattle erythrocytes and causes anaplasmosis. This study reviews diagnostic data of all A. marginale diagnostics performed from 2003 to August 2021 in the Iowa State Veterinary Diagnostic Laboratory. Typically, the referring veterinarian's initial tentative diagnosis was based on presenting clinical signs or necropsy findings. Confirmatory testing at the ISU-VDL consisted of light microscopy evaluation of stained blood smears or molecular diagnostic procedures. A total of 94 cases were submitted with tissue samples from deceased animals, of which 79 were from Iowa and 15 were from other states. The most typical gross lesions were widespread yellow adipose tissue and splenomegaly. Typical histopathological lesions included marked bile stasis and hemosiderin-laden macrophages in the liver and spleen, respectively. Starting in 2013, when PCR was implemented to confirm cases of anaplasmosis, 315/1125 (28%) were positive to A. marginale, and 810 were negative, using a cut-off of 35.0 Ct. The average (±SD) of the positive PCR Ct was 19.5 (±6.0), and the first and third quartiles were 14.9 and 23.4. Most cases occurred between August and November, peaking in September, whether from necropsies or positive blood samples by PCR. The most common tick observed in Iowa, Dermacentor variabilis, is likely the main vector for transmission. Further surveys should be conducted to estimate seroprevalence by geographical location, the density of cattle populations, distribution of known vectors according to season, and strains of A. marginale.

Responses to pathogen exposure in sentinel juvenile fall-run Chinook salmon in the Sacramento River, CA.

This study investigated how the deployment of juvenile Chinook salmon in ambient river conditions and the subsequent exposure to and infection by pathogens was associated with the changes in the expression of genes involved in immune system functioning, general stress and host development. Juvenile fish were deployed in sentinel cages for 21days in the Sacramento River, CA, USA. Gill, kidney and intestinal tissue were sampled at 0, 7, 14 and 21days post-deployment. Pathogen detection and host response were assessed by a combination of molecular and histopathological evaluation. Our findings showed that fish became infected by the parasites Ceratonova shasta, Parvicapsula minibicornis and Ichthyophthirius multifiliis, and to a lesser extent, the bacteria Flavobacterium columnare and Rickettsia-like organisms. Co-infection was common among sentinel fish. Expression of investigated genes was altered following deployment and was often associated with pathogen abundance. This study provides a foundation for future avenues of research investigating pathogens that affect out-migrating Chinook salmon in the Sacramento River, and offers crucial knowledge related to conservation efforts.

Histologic lesions of cestodiasis in octopuses.

Parasitism of cephalopods is common, including infection with Aggregata spp., Ichthyobodo spp., dicyemids, cestodes of the orders Tetraphyllidea and Trypanorhynchidea, and various crustaceans. Cestodiasis in octopuses is reported, although a full histologic description of lesions has not been previously described. Cestodiasis was identified in 10 octopuses of 4 different species, which included 4 common octopuses (Octopus vulgaris), 3 Caribbean reef octopuses (Octopus briareus), 2 two-spot octopuses (Octopus bimaculoides), and 1 giant Pacific octopus (Enteroctopus dofleini). Larval cestodes were present in the cecum (n = 5), intestines (n = 4), digestive gland (n = 3), chitinous alimentary tract (n = 2), renal appendage (n = 1), and salivary duct (n = 1). In 5 cases, larval cestodes invaded tissue and were associated with hemocytic inflammation and tracts of necrotic tissue in the intestines (n = 3), digestive gland (n = 3), and/or renal appendage (n = 1). When present in the chitinous alimentary tract (esophagus, stomach) or cecum, larval cestodes were in the central lumen and not associated with lesions. One adult cestode was identified in the mantle cavity and was not associated with lesions. Other common concurrent parasitic infections included enteric Aggregata spp. infection, branchial Rickettsia-like organism infection, enteric nematodiasis, and an arthropod-associated branchitis.

A health survey of the reef forming scleractinian cold-water corals Lophelia pertusa and Madrepora oculata in a remote submarine canyon on the European continental margin, NE Atlantic

Monitoring of cold-water corals (CWCs) for pathogens and diseases is limited due to the environment, protected nature of the corals and their habitat and as well as the challenging and sampling effort required. It is recognised that environmental factors such as temperature and pH can expedite the ability of pathogens to cause diseases in cold-water corals therefore the characterisation of pathogen diversity, prevalence and associated pathologies is essential. The present study combined histology and polymerase chain reaction (PCR) diagnostic techniques to screen for two significant pathogen groups (bacteria of the genus Vibrio and the protozoan Haplosporidia) in the dominant NE Atlantic deep-water framework corals Lophelia pertusa (13 colonies) and Madrepora oculata (2 colonies) at three sampling locations (canyon head, south branch and the flank) in the Porcupine Bank Canyon (PBC), NE Atlantic. One M. oculata colony and four L. pertusa colonies were collected from both the canyon flank and the south branch whilst five L. pertusa colonies were collected from the canyon head. No pathogens were detected in the M. oculata samples. Neither histology nor PCR detected Vibrio spp. in L. pertusa, although Illumina technology used in this study to profile the CWCs microbiome, detected V. shilonii (0.03%) in a single L. pertusa individual, from the canyon head, that had also been screened in this study. A macroborer was observed at a prevalence of 0.07% at the canyon head only. Rickettsiales-like organisms (RLOs) were visualised with an overall prevalence of 40% and with a low intensity of 1 to 4 (RLO) colonies per individual polyp by histology. L. pertusa from the PBC canyon head had an RLO prevalence of 13.3% with the highest detection of 26.7% recorded in the south branch corals. Similarly, unidentified cells observed in L. pertusa from the south branch (20%) were more common than those observed in L. pertusa from the canyon head (6.7%). No RLOs or unidentified cells were observed in corals from the flank. Mean particulate organic matter concentration is highest in the south branch (2,612 μg l−1) followed by the canyon head (1,065 μg l−1) and lowest at the canyon flank (494 μg l−1). Although the route of pathogen entry and the impact of RLO infection on L. pertusa is unclear, particulate availability and the feeding strategies employed by the scleractinian corals may be influencing their exposure to pathogens. The absence of a pathogen in M. oculata may be attributed to the smaller number of colonies screened or the narrower diet in M. oculata compared to the unrestricted diet exhibited in L. pertusa, if ingestion is a route of entry for pathogen groups. The findings of this study also shed some light on how environmental conditions experienced by deep sea organisms and their life strategies may be limiting pathogen diversity and prevalence.

An Efficient Tetraplex Surveillance Tool for Salmonid Pathogens.

Fish disease surveillance methods can be complicated and time consuming, which limits their value for timely intervention strategies on aquaculture farms. Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and enable high sample throughput with the ability to multiplex several targets using different fluorescent dyes. A ddPCR tetraplex assay was developed for priority salmon diseases for farmers in New Zealand including New Zealand Rickettsia-like organism 1 (NZ-RLO1), NZ-RLO2, Tenacibaculum maritimum, and Yersinia ruckeri. The limit of detection in singleplex and tetraplex assays was reached for most targets at 10−9 ng/μl with, respectively, NZ-RLO1 = 0.931 and 0.14 copies/μl, NZ-RLO2 = 0.162 and 0.21 copies/μl, T. maritimum = 0.345 and 0.93 copies/μl, while the limit of detection for Y. ruckeri was 10−8 with 1.0 copies/μl and 0.7 copies/μl. While specificity of primers was demonstrated in previous studies, we detected cross-reactivity of T. maritimum with some strains of Tenacibaculum dicentrarchi and Y. ruckeri with Serratia liquefaciens, respectively. The tetraplex assay was applied as part of a commercial fish disease surveillance program in New Zealand for 1 year to demonstrate the applicability of tetraplex tools for the salmonid aquaculture industry.

Development and application of a sensitive droplet digital PCR for the detection of red mark syndrome infection in rainbow trout (Oncorhynchus mykiss)

Red Mark Syndrome (RMS) is a skin disease of uncertain etiology affecting farmed rainbow trout (Oncorhynchus mykiss). It consists of single or multiple skin lesions usually localized on the trunk of fish approaching market size.Studies showed as rickettsia-like organism (RLO), currently referred as Midichloria-like organism (MLO), is supposed to be involved in RMS.Midichloria-like has been identified consistently in RMS-affected fish. Its identification within skin and spleen has been possible through the use of PCR methods, including standard PCR, nested PCR and quantitative PCR (qPCR).Droplet digital PCR (ddPCR) is a relatively novel, sensitive, accurate and absolute quantification technique. Recent studies applied successfully this technique to infectious spleen and kidney necrosis virus (ISKNV) in teleosts, proving its superiority when compared to qPCR.The goal of the present study is to establish a sensitive ddPCR method to rapidly detect and quantify MLO DNA.We explored the feasibility of ddPCR to detect MLO from 40 rainbow trout spleen samples and compared the data with qPCR. The detection limit of the ddPCR was found to be 2.25 copy number, which is lower than the 36 copy number determined for TaqMan real-time PCR (qPCR). This indicated that the sensitivity of the ddPCR assay was one order of magnitude higher than the sensitivity of the qPCR assay. The detection results for fish samples showed that the positive detection rate of ddPCR (65%) was higher than that of qPCR (52.5%).The method used has revealed to be specific to MLO and does not cross-react with other fish bacterial DNA. The ddPCR method established in this study shows superiority for detection in minimal volume samples with low bacterial loads and may be used both as surveillance of possible transmission routes and potential sources.

Rickettsia-like and Chlamydia-like organisms of clams and cockles

This datasheet on Rickettsia-like and Chlamydia-like organisms of clams and cockles covers Identity, Associated Diseases, Pests or Pathogens, Hosts/Species Affected.

Black eye syndrome and a systemic rickettsia-like organism in Alaskan Chionoecetes spp. crabs, including normal eyestalk microanatomy.

A black eye syndrome (BES) was discovered in both captive and wild populations of Alaskan snow crabs Chionoecetes opilio and Tanner crabs C. bairdi. Field prevalences ranged from 0.37% (n = 594/161295) to 19.6% (n = 62/316) in snow crabs from the eastern Bering Sea and from 0.09% (n = 15/16638) to 0.7% (n = 133/18473) in Tanner crabs from the same trawl samples, with a slightly greater percentage (1.4%, n = 57/3945) in Tanner crabs from the Aleutian and Kodiak islands fisheries in the Gulf of Alaska. BES is not associated with crab mortality and has 2 distinct manifestations: abnormal black foci of internal eye pigment with no discernible histological lesions, which, in many cases, is followed by corneal shell disease with ulceration and distal eyestalk erosion. It is assumed for this study that these are early and late stages of BES that are somehow related. Our results suggest that early stages of abnormal pigmentation are noninfectious, possibly related to changing ocean conditions affecting crab endocrinology and neuropeptide control of secondary eye pigment. Potential light-induced photoreceptor damage of harvested crabs with dark-adapted eyes is another anthropogenic factor possibly contributing to the early changes in eye pigmentation. Normal eyestalk microanatomy specific for Chionoecetes spp. is provided as necessary baseline information for future studies. Early in the study, an unreported rickettsia-like organism (RLO) was discovered infecting dissected black eyestalks submitted for examination from 5 of 6 dead snow crabs, suggesting association with BES. Subsequent samples indicated the RLO was systemic, infected both black and normal-appearing eyestalks, and was unrelated to BES. However, the multiorgan infection and histopathology indicated the RLO could be a primary pathogen of snow crabs.

A field sentinel study investigating withering syndrome transmission dynamics in California abalones

We examined the risk of withering syndrome (WS) rickettsia-like organism (WS-RLO) infection in sentinel red abalone (Haliotis rufescens) deployed in modules at two Southern California field sites, one adjacent to an abalone farm and one adjacent to wild abalones. WS-RLO DNA was detected in seawater near modules at the wild abalone site but not near the farm (WS-RLO DNA was detected in the farm effluent). More WS-RLO DNA was detected in tissue from abalone near the farm relative to those near wild abalones (p < 0.05). However, infection prevalence and intensity based on histology were low and similar between sites (p > 0.05) and were independent of WS-RLO DNA loads in abalone tissue and seawater. More stippled (ST)-RLO than WS-RLO were observed with more ST-RLO infections near wild abalone than near the abalone farm (p < 0.05). We demonstrate the utility of caged sentinel abalone to better understand pathogen transmission patterns in the field.

An outbreak of crayfish rickettsiosis caused by Coxiella cheraxi in redclaw crayfish (Cherax quadricarinatus) imported to Israel from Australia.

The redclaw crayfish (Cherax quadricarinatus) is a freshwater decapod crustacean, cultured in numerous countries worldwide for both food and ornamental purposes. Redclaw crayfish has become an important aquaculture species due to its physical and biological traits, relatively easy breeding, and a short growing-out period to reach commercial size. Bacterial infections are the second-most studied pathogens of freshwater crayfish. However, redclaw crayfish rickettsiosis, caused by Coxiella cheraxi, was reported in only a few scientific papers in the early 2000s, in Australia and Ecuador. Coxiella cheraxi is a rod-shaped intracellular bacterium that can cause mortality of 22%-80% in naturally infected crayfish. In experimental infections, mortality rates may be even higher (40%-90%). Coxiella cheraxi is closely related to Coxiella burnetii, the agent of Q-fever, which affects ruminants (goats, sheep, and cattle) and occasionally may cause zoonotic infections. According to the scientific knowledge available, C. cheraxi is a species-specific pathogen because it has been only detected in Cherax quadricarinatus and thus far, there is no evidence of a zoonotic potential. In this study, we describe an outbreak of rickettsiosis in a batch of redclaw crayfish imported to Israel from an Australian hatchery, observed 2 months after introduction in a quarantine facility. Initial mortality was evaluated through histopathology, revealing infection by rickettsia-like organisms (RLO) that were subsequently investigated by molecular analysis and transmission electron microscopy examination. Phylogenetic analysis revealed that the detected RLO were closely related to C. cheraxi from a single source (Australian strain TO98), available in free publicly accessible databases. After 5 months in quarantine, almost 99% of the crayfish population had died. Our findings raise valuable questions related to aquatic animal trade and the importance of mitigation measures, such as quarantine and routine diagnostic procedures, to limit the spread of infectious diseases.

Intracellular bacteria in New Zealand shellfish are identified as Endozoicomonas species.

Kaimoana (shellfish, seafood) is an important food source and a significant social and cultural component of many New Zealand communities, especially the indigenous Māori. Over the past decade a decline has been detected in shellfish health and an increase in mortality events around New Zealand. Intracellular bacteria termed Rickettsia-like organisms (RLOs) have been observed in New Zealand bivalve molluscs during shellfish mortality events. Affected bivalves include cockles Austrovenus stutchburyi, ringed dosinia Dosinia anus, green-lipped mussels Perna canaliculus, pipi Paphies australis, toheroa Paphies ventricosa, tuatua Paphies subtriangulata, deepwater tuatua Paphies donacina and scallops Pecten novaezelandiae. RLOs are an informal morphology-based classification of intracellular bacteria, with the exact identification often unknown. Using shellfish collected during mortality events from 2014 to 2019 and apparently healthy samples collected in 2018 and 2019, we aimed to identify RLOs in New Zealand shellfish. Bacterial 16S rRNA gene sequences from RLO-infected shellfish showed >95% identity to published Endozoicomonas species. In situ hybridization confirmed the presence of the sequenced gene in the gill epithelium and digestive epithelium of all study species. A genus-specific quantitative PCR, targeting the 16S rRNA gene was developed to detect Endozoicomonas spp. in shellfish tissue. Prevalence of Endozoicomonas spp. in samples from mortality events and healthy shellfish analysed by quantitative PCR was high. Samples collected from mortality events, however, had a significantly higher load of Endozoicomonas spp. than the healthy samples. These results give us a greater understanding of these intracellular bacteria and their presence in populations of New Zealand shellfish.

Characterisation and distribution of the bacterial genus Endozoicomonas in a threatened surf clam.

The toheroa Paphies ventricosa is a large Aotearoa New Zealand (ANZ) endemic surf clam of cultural importance to many Māori, the Indigenous people of ANZ. Extensive commercial and recreational harvesting in the 20th century dramatically reduced populations, leading to the collapse and closure of the fishery. Despite being protected for >40 yr, toheroa have inexplicably failed to recover. In 2017, intracellular microcolonies (IMCs) of bacteria were detected in 'sick' toheroa in northern ANZ. Numerous mass mortality events (MMEs) have recently been recorded in ANZ shellfish, with many events linked by the presence of IMCs resembling Rickettsia-like organisms (RLOs). While similar IMCs have been implicated in MMEs in surf clams elsewhere, the impact of these IMCs on the health or recovery of toheroa is unknown. A critical first step towards understanding the significance of a pathogen in a host population is pathogen identification and characterisation. To begin this process, we examined 16S rRNA gene sequences of the putative IMCs from 4 toheroa populations that showed 97% homology to Endozoicomonas spp. sequences held in GenBank. Phylogenetic analysis identified closely related Endozoicomonas strains from the North and South Island, ANZ, and in situ hybridization, using 16S rRNA gene probes, confirmed the presence of the sequenced IMC gene in the gill and digestive gland tissues of toheroa. Quantitative PCR revealed site-specific and seasonal abundance patterns of Endozoicomonas spp. in toheroa populations. Although implicated in disease outbreaks elsewhere, the role of Endozoicomonas spp. within the ANZ shellfish mortality landscape remains uncertain.

High-Quality Draft Genome Sequence of a Rickettsiales Bacterium Found in Acropora tenuis Coral from Okinawa, Japan.

Rickettsiales-like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of Rickettsiales bacterial strain SESOKO1, found in Acropora tenuis coral, using single-cell genome technology.

Coexistence of the stippled Rickettsiales-like prokaryote with Candidatus Xenohaliotis californiensis and the bacteriophage pCXc in farmed red abalone Haliotis rufescens from Mexico

Candidatus Xenohaliotis californiensis (CXc) is a Rickettsiales-like organism (RLO) that is considered the causal agent of withering syndrome (WS), a chronic and potentially fatal disease for several abalone species. CXc can be infected by a bacteriophage (pCXc), and both phage-infected and non-phage-infected CXc can coexist with a presumable different species of RLO tentatively named stippled Rickettsiales-like organism (ST-RLO). Currently, the taxonomic affiliation of the ST-RLO is unknown, and no studies have been conducted to determine its presence in infected abalones in the Peninsula of Baja California, Mexico. To determine whether the ST-RLO is a histological variant of CXc or a new species of RLO, we performed histology and species-specific in situ hybridization (ISH) for CXc and pCXc in red abalone, Haliotis rufescens, infected with CXc and hyperparasitized by pCXc, which also showed the presence of the ST-RLO. Additionally, a histological analysis of the presence of all of these RLOs from 2008 to 2019 from an aquaculture facility was performed. The results showed that all ST-RLOs were negative by ISH for CXc and pCXc, suggesting that the ST-RLO is a different species of bacterium and that it is not susceptible to infection by pCXc. Histologically similar alterations to those produced by CXc in the post esophagus were observed. The three RLOs regularly coexist in all the infected abalones, and there is a relatively constant proportion of each kind of RLO over time, suggesting that equilibrium is reached during coexistence; the largest percentage corresponded to CXc non phage infected (81%), followed by CXc infected by pCXc (16%) and the ST-RLO (3%). In all cases, the ST-RLO infection remained at a subclinical level making it difficult to determine if it acts as pathogen or as a commensal. Therefore, the genetic characterization of the ST-RLO and pathogenicity studies are needed to further elucidate the role of this new RLO in the development of of WS in abalone species of North America.

"Candidatus Trichorickettsia mobilis", a Rickettsiales bacterium, can be transiently transferred from the unicellular eukaryote Paramecium to the planarian Dugesia japonica.

Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont “Candidatus (Ca.) Trichorickettsia mobilis” was identified in the macronucleus of the ciliate Paramecium multimicronucleatum. We performed transfection experiments of this RLO to planarians (Dugesia japonica) per os. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians’ enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by Rickettsiales or other RLOs and occurring in fish farms or in the wild.

Ecology, histopathology, and microbial ecology of a white-band disease outbreak in the threatened staghorn coral Acropora cervicornis.

This study is a multi-pronged description of a temperature-induced outbreak of white-band disease (WBD) that occurred in Acropora cervicornis off northern Miami Beach, Florida (USA), from July to October 2014. We describe the ecology of the disease and examine diseased corals using both histopathology and next-generation bacterial 16S gene sequencing, making it possible to better understand the effect this disease has on the coral holobiont, and to address some of the seeming contradictions among previous studies of WBD that employed either a purely histological or molecular approach. The outbreak began in July 2014, as sea surface temperatures reached 29°C, and peaked in mid-September, a month after the sea surface temperature maximum. The microscopic anatomy of apparently healthy portions of colonies displaying active disease signs appeared normal except for some tissue atrophy and dissociation of mesenterial filaments deep within the branch. Structural changes were more pronounced in visibly diseased fragments, with atrophy, necrosis, and lysing of surface and basal body wall and polyp structures at the tissue-loss margin. The only bacteria evident microscopically in both diseased and apparently healthy tissues with Giemsa staining was a Rickettsiales-like organism (RLO) occupying mucocytes. Sequencing also identified bacteria belonging to the order Rickettsiales in all fragments. When compared to apparently healthy fragments, diseased fragments had more diverse bacterial communities made up of many previously suggested potential primary pathogens and secondary (opportunistic) colonizers. Interactions between elevated seawater temperatures, the coral host, and pathogenic members of the diseased microbiome all contribute to the coral displaying signs of WBD.

First record of new rickettsia-like organism in the blue king crab Paralithodes platypus from the Sea of Okhotsk: Distribution, morphological evidence and genetic analysis

An infection caused by a rickettsia-like organism (RLO) was detected in the blue king crab Paralithodes platypus from the eastern Sea of Okhotsk. The external signs of the disease are lethargy and an empty gastrointestinal tract. Dissection of infected individuals revealed that their hepatopancreas was light yellow in color. The causative agent of infection is Gram-negative rod-shaped bacterium targeted exclusively at hepatopancreas tissues. In the cytoplasm of infected cells, the bacteria are enclosed in parasite vacuoles or located immediately in cytosol. An ultrastructural analysis showed two main morphological types corresponding to the life cycle stages in the RLO: the vegetative stage of intermediate bodies, characterized by growth and division processes, and the infection stage of elementary bodies, which are spore-like non-dividing short rods surrounded by a multilayered membrane and having an osmiophilic inclusion body. At the terminal stage of infection, as a result of lysis of the infected cells, the RLO enters the lumen of the hepatopancreatic tubules which contributes to the spread of infection. According to genetic analysis based on 16S rRNA gene sequences, the RLO from P. platypus is most closely related to the Candidatus Hepatobacter penaei, NCBI #JX981946 (94.7% similarity) and NCBI #KY363553 (94.1% similarity). The high level of genetic differences (more than 5%) of the studied pathogen, along with the structural features, allows characterizing the RLO isolated from P. platypus as a new species of the genus Candidatus Hepatobacter paralithodi nov. sp., NCBI #MK928971.