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Rickettsia-like organisms (RLO) are important pathogens of many aquatic animals; they are obligate intracellular bacteria (Garrity et al. 2006). Rickettsia-like organisms have been found in crustaceans, fish, gastropods, and bivalves (Cvitanich et al. 1991, Bower 2000, Wang & Gu 2002), causing tissue damage and pathological alterations, with a negative impact on aquaculture (Wu et al. 2005). The oyster Crassostrea gigas (Thunberg, 1793) has great commercial importance in countries such as the United States, Japan, China, Korea, and Taiwan (Chavez-Villalba 2014). It has been reported that rickettsial infections can cause mass mortalities in C. gigas culture (Renault & Cochennec 1995, Wu & Pan 2000). Rickettsia-like organism colonies in marine bivalves have usually been reported to be more common in gill tissues (Sun & Wu 2004), but in C. gigas and Crassostrea rhizophorae (Guilding, 1828) damage has been associated with basophilic RLO in the epithelia of digestive tubules and stomach (Comps et al. 1979, Sabry et al. 2011, Sabry et al. 2013). Basophilic is a technical term used by cytologists and histologists that describes the microscopic appearance of cells and tissues, after they are stained with a basic dye such as the crystal violet dye used to stain bacteria or hematoxylin used for tissues, and corresponds to a dark blue or purple stain (basic/positive). Eosinophilic or acidophilic describes the appearance of cells and structures after being stained in red or pink (acid/positive) by an acid dye such as eosin. Gulka and Chang (1984) indicated that in most of the cases, Rickettsiales do not cause high mortalities, but provoke only slight damage in the tissues. Similarly, Romalde and Barja (2010) mentioned that there is no clear association between mass mortalities of C. gigas and the presence of Rickettsia.

Most of the RLO reported in oysters and other bivalves have a basophilic affinity (Comps et al. 1979, Morrison & Shum 1983, Elston 1986, Fries & Grant 1991, Fryer & Lannan 1994, Renault & Cochennec 1995, Azevedo et al. 2005, Miossec et al. 2009, Romalde & Barja 2010, Watanabe et al. 2015). Acidophilic RLO have also been observed in some bivalves (Meyers 1981, Wu & Pan 1999, Zhu et al. 2012, Azmi et al. 2014) such as Crassostrea rivularis (Gold, 1861) (Zhang et al. 2017). Both basophilic RLO and E-RLO inclusions are classified as belonging to the family gammaproteobacteria (Zhang et al. 2017).

Correct identification of an infectious agent is essential to control and prevent the spread of the pathogen. Because Rickettsia is an intracellular pathogen, in vitro culture (typically cell culture) is complex (Fryer & Lannan 1994). Although detection of RLO with traditional histology is feasible, molecular methods are more reliable. Since 1997, significant mortalities of oysters have been observed in Mexico and have continued until now with different degrees of severity (Chavez-Villalba 2014). Although several pathogens capable of causing mass mortalities have been identified, studies have not been conclusive regarding the causative agent (Chavez-Villalba 2014). The interaction of infectious agents with their hosts is a complex interplay, requiring detailed knowledge for their prevention and control. To provide additional information about a potential pathogen, this study describes, for the first time, colonial growth [i.e., growth of bacteria in both size and number in the parasitophorous vacuole of the infected cell, according to Sun and Wu (2004)] and development of an E-RLO in some tissues of Crassostrea gigas.


Histopathology Inspection

Seven sick (with mantle blisters and gill damage) and three healthy Crassostrea gigas adult oysters were collected in Laguna de San Ignacio, Baja California Sur, Mexico, in August 2014. Cross sections of the mantle, gill, digestive tract, and gonad were prepared from each animal. Tissues were fixed with Davidson's solution, dehydrated, and embedded in paraffin. Sections of 4 [micro]m were obtained and stained with hematoxylin and eosin (H&E) (Humason 1979) and Giemsa (Lillie 1965). In addition, serial histological sections were prepared for in situ hybridization (ISH). Three sections from each oyster were photographed with a digital camera, using an optical microscope (Olympus BX41), and processed with the Image-Pro Premier (v 9.0) Olympus-Media Cybernetics software to calculate the area infected by E-RLO in the mantle epithelium (Venkateswaran et al. 2014). Hematoxylin binds to basophilic substances and they are dyed violet-blue (such as heterochromatin and ribosomal RNA, which are acidic and negatively charged); eosin binds to acidophilic substances with positive charge and they are dyed pink or orange (such as cationic amino acids and proteins). The intensity of the infection was obtained with the following equation:

Infection intensity = area occupied by E - RLO X 100/epithelial total area.

Molecular Identification

Extraction of genomic DNA was performed from 100 mg of mantle tissue by the salting-out method (Miller et al. 1988). The samples were incubated in lysis buffer (50 mM Tris, pH 8.0; 100 mM NaCl; 100 mM ethylenediaminetetraacetic acid, pH 8.0; and 1% sodium dodecyl sulfate) and proteinase K (Promega) for 20 min at 65[degrees]C in a thermal bath. After incubation, the nucleoproteins were separated with 6 M NaCl and DNA was precipitated with absolute ethanol. The ethanol was removed and the pellet was dried in an oven at 30[degrees]C for 3 min. The DNA pellet was resuspended with molecular grade water (Sigma-Aldrich). DNA concentration and purity were verified by using a Nanodrop 2000 (Thermo). The DNA was stored at -20[degrees]C until use. Detection by polymerase chain reaction (PCR) was performed using the primers RLO-F: 5-GTGGGGAGCAAACAGGATA-3 and RLO-R: 5-CACCGG CAGTCTCCTTAGAG-3. These primers were designed based on 16S RNA sequences from Oceanrickettsia ariakensis (accession numbers DQ123914.1 and DQ118733.1). Sequence alignment was performed using MEGA 4 software (Tamura et al. 2007). The primer was designed using online software Primer 3 (Untergasser et al. 2012). The primers were tested in silico to avoid primer-dimer formation by using a multiple primer analyzer (Thermo Scientific Web Tools) and tested for specificity by using NCBI BLAST (Altschul et al. 1997).

The PCR mixture was prepared as follows: 100 ng of DNA, 1X PCR buffer, 1.5 mM Mg[Cl.sub.2], 200 [micro]M of each dNTP, 0.4 [micro]M of each primer, and 0.5 U of Taq polymerase GoTaq Flexi (Promega) in a final volume of 12.5 [micro]L. Polymerase chain reaction amplification conditions were as follows: 94[degrees]C for 3 min, 35 cycles of 94[degrees]C for 1 min, 55[degrees]C for 1 min, and 72[degrees]C for 1 min. As it was not possible to use a true positive control of Ocean-rickettsia ariakensis, it was replaced by the purified fragment obtained from samples of Crassostrea gigas identified histologically as positive for E-RLO. Polymerase chain reaction water was used as negative control.

Polymerase chain reaction products were visualized in 1.5% agarose gel. The gels were stained with ethidium bromide at 1 [micro]g/mL and visualized by ultraviolet illumination. The molecular weight marker PhiX174 DNA HaeIII (Promega) was used as a reference. All the fragments were sent to the University of Arizona Genetics Core for bidirectional sequencing. The sequences were edited using the SEQUENCHER program (Sequencher 4.7; Gene Codes Corporation, Ann Arbor, MI) and aligned using the MEGA 4 program, ClustalW option (Tamura et al. 2007). The obtained sequences were submitted to the GenBank database using the BLAST algorithm (Madden 2002).

For ISH, the digoxigenin (DIG) DNA labeling and detection kit (Roche Molecular Biochemicals) was used to label the RLO PCR product by random priming with DIG-dUTP, and the detection was performed with an anti-DIG antibody coupled to alkaline phosphatase, followed by incubation in NBT/BCIP. The tissues were counterstained with Bismarck brown, examined, and photographed with an optical microscope (Olympus BX41) with a digital camera (Nikon Digital Sight DS-Ri1).

Statistical Analysis

Data normality was analyzed with the Kolmogorov-Smirnov test and Lilliefors tests and the variance homogeneity was analyzed with Bartlett's test. Percentage data were normalized by an arcsine transformation. For data comparison, Student's t-test was performed to determine the infection intensity in the mantle between PCR-positive and negative organisms.


Histopathologcal Inspection

Histological examination showed the presence of intraepithelial aggregations of RLO, forming intensely stained inclusions with eosinophilic (acidophilic) affinity. The individual size of the Rickettsia was too small to be measured individually by optical microscopy. Different stages of E-RLO development were observed, with variable sizes and shapes within infected epithelial cells. The E-RLO growth started with focal eosinophilic small and compact granules alone or grouped with up to four granules with a diameter of 1.4-2.9 [micro]m each (Fig. 1A); at this stage, no parasitophorous vacuoles have yet been formed. In the next stage, the granules increased in number inside the cells, forming parasitophorous vacuoles (containing compact colonies), like intracellular inclusions, with circular or ovoid forms (Fig. 1B, C). Also, chain forms in the epithelia of the digestive tract and palps were observed; in this stage, the parasitophorous vacuoles (acidophilic) were of great size with many acidophilic granules. In an advanced development of E-RLO growth, the presence of many corpuscles (smaller than granules) inside the parasitophorous vacuoles (moderately eosinophilic) and few colonies were observed (Fig. 1D). In this stage, there was an intense cellular infection by E-RLO, with lysis of some host cells, and release of corpuscles able to infect new cells (Fig. 1D).

Intracellular inclusions (parasitophorous vacuoles) in the epithelia of the mantle, gills, labial palps, and digestive tract were observed; these tissues were different in respect of uninfected tissues (negative control) on cellular appearance (Fig. 2A-H). Only in one oyster E-RLO was detected in the epithelium of the digestive gland. The increase in parasitophorous vacuoles containing increased numbers and sizes of bacteria/granules within the vacuoles resulted in moderate hypertrophy of the infected cell (Fig. 2D).

Molecular Identification

Detection by PCR confirmed the presence of RLO. A DNA fragment of 398 bp was amplified in seven apparently sick oysters (Fig. 3). The sequenced products had a similarity of 99% to Oceanrickettsia ariakensis (accession number DQ123914), 98% for unidentified bacteria, 94% for uncultured rickettsia, and 89% for the genus Rickettsia according to Blast results.

In situ hybridization confirmed the molecular and histological diagnosis, as E-RLO was found in mantle tissues, gills, labial palps, the digestive tract, and male and female gametes. The hybridization signal (gray colonies) was detected in all positive controls and was found in mantle tissues, gills, labial palps, the digestive tract, and male and female gametes, but the signal was not detected in the negative control (Fig. 4A-F). The epithelia with high numbers of cells infected with E-RLO caused necrosis (Fig. 4C).

All organisms analyzed by histology and ISH showed E-RLO in the mantle, but only 70% of the oysters were positive for the presence of E-RLO by the PCR method. Infection intensity in the mantle of PCR-positive oysters (12.4%, P< 0.05) was statistically higher than that of PCR-negative organisms (2.2%, P < 0.05).


Most of the research on Rickettsia (or RLO) in bivalves describes intracellular, basophilic inclusions (microcolonies), not associated with severe tissue damage (Comps et al. 1979, Morrison & Shum 1983, Elston 1986, Fries & Grant 1991, Renault & Cochennec 1995, Azevedo et al. 2005, Miossec et al. 2009, Romalde & Barja 2010). Eosinophilic Rickettsia-like organisms were found in Crassostrea gigas, capable of causing damage to the mantle epithelium, gills, labial palps, digestive tract, digestive gland, and possibly gametes (female and male). To date, only few studies have found eosinophilic Rickettsia to be associated with pathological alterations in bivalves (Meyers 1981, Wu & Pan 1999, Zhu et al. 2012, Azmi et al. 2014). In Crassostrea oysters, there is only one investigation reporting eosinophilic Rickettsia in Crassostrea ariakensis and Crassostrea rivularis (cultured in China), with the ability to destroy the gill tissue, causing the death of organisms (Wu & Pan 2000, Wu et al. 2005). Basophilia and acidophilia depend on the amount of ribosomes in the cytoplasm; for this reason, cells with few ribosomes are acidophilic or eosinophilic (because of their affinity to eosin), and cells with many ribosomes are basophilic (Kuhnel 2005). It has been reported that basophilic species such as Rickettsia prowazekii have more ribosomes than other bacteria, which is a characteristic of the species (Pang & Winkler 1994, Winkler 1995). Based on this, it is possible that the E-RLO found in this work is a different species from those reported in other investigations of C. gigas.

Although other works have mentioned the presence of granules (acidophiles) within parasitophorous vacuoles of several sizes in acidophilic RLO, they do not describe the sizes of the inclusions inside the parasitophorous vacuoles of infected cells (Meyers 1981, Wu & Pan 1999, Zhu et al. 2012, Azmi et al. 2014). Various forms and sizes of inclusions were found inside the parasitophorous vacuoles of infected cells, resulting from the different stages of colonial development of E-RLO. These stages are as follows: (1) Small colonies (acidophilic granules) inside host cells are formed. (2) The clusters of such granules are wrapped, forming a parasitophorous vacuole which increases in size with the increase in the number of intravacuolar granules. (3) The stage of propagation starts with the host cell lysis (heavily infected) and release of E-RLO corpuscles that are able to infect new cells. It has been observed that bacterial growth and development depend on the phylogenetic group to which the bacterium belongs (Ueda et al. 2004). This confirms that the RLO found in this work is acidophilic and has characteristics of colonial growth and development similar to other Rickettsiales (Bastos et al. 2009, Friche-Passos 2012), which was also confirmed by PCR and ISH.

Histopathological methods are very informative regarding disease evolution. Sometimes the method has limitations for the diagnosis of epizootics; therefore, additional methods such as PCR and ISH are necessary to confirm diagnosis (Culloty & Mulcahy 2007). In this work, histopathology was useful to identify tissue damage caused by the E-RLO, but not for the RLO detection in the gametes. In bivalves, the oocytes show acidophilic staining (H&E) in different stages of development; for this reason, E-RLO colonies are difficult to distinguish by conventional histology. One factor that may favor the pathogen virulence is its route of transmission. In the marine environment, horizontal transmission may occur by ingestion of water and food with pathogens or by direct contact with diseased organisms. Another route is vertical transmission, which occurs by the fertilization of gametes carrying the pathogen (Le Gall et al. 1991). In marine bivalves, Rickettsiae have not been observed in the gametes, ruling out vertical transmission (Gulka et al. 1983). For instance, Le Gall et al. (1991), using serological analysis, found that Pecten maximus juveniles obtained from Rickettsiales-infected parents were consistently negative to the presence of Rickettsia. Nevertheless, Meyers and Burton (2009) pointed out that the routes of transmission of marine Rickettsiales are not well known. In this study, ISH demonstrated the presence of E-RLO in both gametes, suggesting the transmission of E-RLO to the larvae. Although it is important to consider that the process of atresia (possibly due to the RLO) might decrease the fertilization of infected gametes, in male gametes infected with RLO, alterations were not observed. It is important to carry out further studies to evaluate the viability of the gametes infected with the E-RLO to establish if vertical transmission is achieved in Crassostrea gigas.

The results of this study provide valuable information on the development of Rickettsia with characteristics different from those reported in other works on Crassostrea gigas, which can cause tissue damage and infect the gametes. Further work will be required to define the species or to establish it as a new species. In addition, carrying out studies on the transmission routes will be very useful to establish sanitary control measures related to this pathogen.


The authors thank Nairoby Pacheco Carlon for samples processing and E. Meza Chavez. from the histology laboratory at CIBNOR for histopathology specimen processing. This work was financed and undertaken as part of the PROINNOVA-CONACyT 212952 project.


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(1) Centro de Investigationes Biologicas del Noroeste, Instituto Politecnico National 195, Playa Palo de Santa Rita Sur, La Paz C.P. 23096, B.C.S. Mexico; (2) Centro de Investigation en Alimentation y Desarrollo, A.C. Unidad Mazatlan, Av. Sabalo-Cerritos s/n, Estero del Yugo, Mazatldn, Sinaloa C.P. 82000, Mexico

(*) Corresponding author. E-mail:

DOI: 10.2983/035.037.0306
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Author:Carvalho-Saucedo, Liliana; Dimitrov, Ilie Sava Racotta; Jaramillo, Carmen Rodriguez; Garcia-Gasca, A
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1MEX
Date:Aug 1, 2018

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