Laser Capture Microdissection of Vibrio splendidus from Haliotis rufescens during a post-spawning mortality episode, an alternative method for the study of diseases in aquaculture

Autores/as

Rebeca Vásquez-Yeomans Centro de Investigación Científica y de Educación Superior de Ensenada, (CICESE) Km 107 Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860. Ensenada, Baja California, México
Roberto Cruz-Flores Centro de Investigación Científica y de Educación Superior de Ensenada, (CICESE) Km 107 Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860. Ensenada, Baja California, México
Jorge Cáceres-Martínez Centro de Investigación Científica y de Educación Superior de Ensenada, (CICESE) Km 107 Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860. Ensenada, Baja California, México

Palabras clave:

Histopatología, Abulón rojo, Vibrio splendidus, Microdisección láser

Resumen

Histopathological examination allows direct observation of tissue structure, the presence of pathogens, and their association with cellular and tissue alterations; however, it is almost impossible to isolate and identify these microorganisms from histological preparations. The advent of laser capture microdissection (LCM) technologies in conjunction with molecular tools is currently being used for genomic, transcriptomic, and proteomic studies in human and veterinary pathology and parasitology. A combination of these techniques may be used to identify a particular pathogen. However, this innovative approach has rarely been used to study pathogens in aquatic organisms. Clinical, histological, LCM, PCR, Sanger sequencing, and conventional bacteriological analysis of red abalones, Haliotis rufescens, from a broodstock suffering from a post-spawning mortality event were carried out. In all cases, the clinical analysis showed swelling of muscle tissues, visceral mass and excessive mucus production. The histopathological analyses showed curved rod-shaped bacteria affecting the muscle tissues around the mouth, head, foot muscle, and endothelium of haemolymphatic vessels. Analysis by LCM, PCR, and Sanger sequencing allowed the bacteria in the affected tissues to be identified as Vibrio splendidus. On the other hand, conventional bacteriological and molecular analysis of a subsample of the head and muscle of one of these abalones allowed the following species to be isolated and identified: V. splendidus, V. chagasii, V. mediterranei, Vibrio sp., and Pseudoalteromonas sp. LCM technology allowed the detection of the bacterial species proliferating in the tissues and allowed their differentiation from those isolated from whole fresh tissues. This combination of modern techniques helps to define which bacteria should be selected to carry out Koch's postulates and to establish their role in the development of a disease and/or mortality event. The results suggest a detailed study on the role of V. splendidus in post-spawning mortality events in H. rufescens.

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Allam B., Paillard C., Ford S. E. (2002). Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams. Dis. Aquat. Org. 48:221-223.

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215:403-410. https://10.1016/S0022-2836(05)80360-2.Bogaert L., Martens A., Kast W.M., Van Marck E., De Cock H. (2010). Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors. Vet. Microbiol. 146:269-275.

Cáceres-Martínez J., Vásquez-Yeomans R., Danigo P., Reyes-Roel C. (2017). Histological alterations in oysters Crassostrea gigas that survived a summer mortality event in Baja California, Mexico. J. Aquat. Anim. Health 30:31-38. https://doi.org/10.1002/aah.10006.

Cardinaud M., Barbou A., Capitaine C., Bidault A., Dujon A. M., Moraga D., Paillard C. (2014). Vibrio harveyi adheres to and penetrates tissues of the European abalone Haliotis tuberculata within the first hours of contact. Appl. Environ. Microbiol. 80:6328–6333. https://doi.org/10.1128/AEM.01036-14.

Chankeaw W., Lignier S., Richard C., Ntallaris T., Raliou M., Guo Y., Plassard D., Bevilacqua C., Sandra O., Andersson G., Humblot P., Charpigny G. (2021). Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells. BMC Genomics 22:451. https://doi.org/10.1186/s12864-021-07712-0.

Cheney D., MacDonald B., Elston R. (2000). Summer mortality of Pacific oysters, Crassostrea gigas (Thunberg): initial findings on multiple environmental stressors in Puget Sound, Washington, 1998. J. Shellfish Res. 19:353–359.

Cruz-Flores R., Cáceres-Martínez J., Vásquez-Yeomans R. (2015). A novel method for separation of Rickettsiales-like organism “Candidatus Xenohaliotis californiensis” from host abalone tissue. J. Microbiol. Methods 115:79-82.

Cruz-Flores R., López-Carvallo J. A., Cáceres-Martínez J., Dhar A. K. (2022). Microbiome analysis from formalin-fixed paraffin-embedded tissues: Current challenges and future perspectives. J. Microbiol. Methods 196:106476

Delaporte M., Soudant P., Lambert C., Jegaden M., Moal J., Pouvreaud S., Dégremont L., Boudry P., Samain J.-F. (2007). Characterization of physiological and immunological differences between Pacific oyster (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions. J. Exp. Mar. Biol. Ecol. 353:45-57.

Friedman C. S., Estes R. M., Stokes N. A., Burge C. A., Hargove J. S., Barber B. J., Elston R. A., Burreson E. M., Reece K. S. (2005). Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes. Dis. Aquat. Org. 63:33–41.

Friedman C. S., Crosson L. M. (2012). Putative phage hyperparasite in the Rickettsial pathogen of Abalone, “Candidatus Xenohaliotis californiensis.” Microb. Ecol. 64:1064–1072. https://doi.org/10.1007/s00248-012-0080-4.

Garnier M., Labreuche Y., Garcia C., Robert M., Nicolas J. L. (2007). Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas. Microb. Ecol. 53:187-196.

Guisande J. A., Montes M., Farto R., Armada S. P., Perez M. J., Nieto T.P. (2004). A set of tests for the phenotypic identification of culturable bacteria associated with Galician bivalve mollusc production. J. Shellfish Res. 23:599.

Guo W., Hu Y., Qian J., Zhu L., Cheng, J., Liao J., Fan X. (2023). Laser capture microdissection for biomedical research: towards high-throughput, multi-omics, and single-cell resolution. J. Genet. and Genomics 50:641-651.

Göcz B., Rumpler É., Szentkirályi-Tóth S., Skrapits K., Takács S., Sárvári M., Farkas I., Póliska S., Hrabovszky E. (2025). Laser-capture microdissection for spatial transcriptomics of immunohistochemically detected neurons. J. Biol. Chemistry. https://doi.org/10.1016/j.jbc.2024.108150.

Handlinger J., Carson J., Donachie L., Gabor L., Taylor D. (2005). Bacterial infection in Tasmanian farmed abalone: causes, pathology, farm factors and control options. In: Walker P. J., Lester R. G., Bondad-Reantaso M. G. (eds.). Diseases in Asian Aquaculture V. Proceedings of the 5th Symposium on Diseases in Asian Aquaculture. Fish Health Section, Asian Fisheries Society, Manila. pp. 289-299.

Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., Williams S. T. (1994). Bergey’s Manual of Determinative Bacteriology (Ninth Edition). Williams & Wilkins. Baltimore.

Howard D. W., Smith C. S. (2004). Histological techniques for marine bivalve mollusks. NOAA Technical Memorandum NMFS-F/NEC-25. Woods Hole, MA: U.S. Department of Commerce.

Ina-Salwany M. Y., Al-Saari N., Mohamad A., Mursidi F. A., Mohd-Aris A., Amal M. N. A., Kasai H., Mino S., Sawabe T., Zamri-Saad M. (2019). Vibriosis in Fish: A Review on Disease Development and Prevention. J. Aquat. Anim. Health 31:3-22. https://doi.org/10.1002/aah.10045.

Kim M., Chun J. (2014). 16S rRNA Gene-Based Identification of Bacteria and Archaea using the EzTaxon Server. In Modal Sosial Dalam Pendidikan Berkualitas Di Sekolah Dasar Muhammadiyyah Muitihan (Issue September, pp. 61–74). https://doi.org/10.1016/bs.mim.2014.08.001

Klitgaard K., Mølbak L., Jensen T. K., Lindboe C. F., Boye M. (2005). Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization. BioTechniques 39:864-868. https://doi.org/10.2144/000112024.

Li Y., Qin J. G., Li X., Benkendorff K. (2009). Spawning-dependent stress response to food deprivation in Pacific oyster, Crassostrea gigas. Aquaculture 286:309-317. http://dx.doi.org/10.1016/j.aquaculture.2008.09.035.

Li Y., Qin J. G., Li X., Benkendorff K. (2010). Assessment of metabolic and immune changes in postspawning Pacific oyster Crassostrea gigas: identification of a critical period of vulnerability after spawning. Aquac. Res. 41:155-165.

Liu A. (2010). Laser capture microdissection in the tissue biorepository. J. Biomol. Tech. 21:120-125.

Lu J., Kwan B. C., Lai F. M., Tam L. S., Li E. K., Chow K. M., Wang G., Li P. K., Szeto C. C. (2012). Glomerular and tubulointerstitial miR-638, miR-198 and miR-146a expression in lupus nephritis. Nephrology 17:346-351. http://dx.doi.org/10.1111/j.1440-1797.2012.01573.x.

Marcu A., Nitusca D., Vaduva A., Baderca F., Cireap N., Coricovac D., Dehelean C. A., Seclaman E., Ilina R., Marian C., (2021). Long non-coding RNA expression in laser micro-dissected luminal A and triple negative breast cancer tissue samples-a pilot study. Medicina 57:371. http://dx.doi.org/10.3390/medicina57040371.

Morton M. L., Bai X., Merry C. R., Linden P. A., Khalil A. M., Leider R. S., Thompson C. L. (2014). Identification of mRNAs and LincRNAs associated with lung cancer progression using nest-generation RNA sequencing from laser microdissected archival FFPE tissue specimens. Lung Cancer 85:31-39.

Nicolas J. L., Basuyaux O., Mazurié J., Thébault A. (2002). Vibrio carchariae, a pathogen of the abalone Haliotis tuberculata. Dis. Aquat. Org. 50:35-43.

Okuducu A. F., Hahne J. C., Von Deimling A., Wernert N. (2005). Laser-assisted microdissection, techniques and applications in pathology (Review). Int. J. Mol. Med. 15:763-769. https://doi.org/10.3892/ijmm.15.5.763.

Perdue J. A., Beattie J. H., Chew K. K. (1981). Some relationship between gametogenic cycle and summer mortality phenomenon in the pacific oyster (Crassostrea gigas) in Washington State. J. Shellfish Res. 1:9-16.

Pujalte M. J, Garay E. (1986). Proposal of Vibrio mediterranei sp. nov.: A new marine member of the genus Vibrio. Int. J. Syst. Bacteriol. 36:278-281.

Samain J. F., Dégremont L., Soletchnik P., Haure J., Bédier E., Ropert M., Moal J., Huvet A., Bacca H., Van Wormhoudt A., Delaporte M., Costil K., Pouvreau S., Lambert C., Boulo V., Soudant P., Nicolas J. L., Le Roux F., Renault T., Gagnaire B., Geret F., Boutet I., Burgeot T., Boudry P. (2007). Genetically based resistance to summer mortality in the Pacific oyster (Crassostrea gigas) and its relationship with physiological, immunological characteristics and infection processes. Aquaculture 268:227-243.

Sambrook J., Russell D. W. (2001). Molecular Cloning: A Laboratory Manual. 3rd Edition, Vol. 1, Cold Spring Harbor Laboratory Press, New York.

Sawabe T. 2006. The mutual partnership between Vibrio halioticoli and abalones. In: Thompson F.L., Austin B. and Swings J. (Eds.), The biology of vibrios. Wiley, pp. 219-230. https://doi.org/10.1128/9781555815714

Searcy-Bernal R., Ramade-Villanueva M., Altamira B. (2010). Current status of abalone fisheries and culture in Mexico. J. Shellfish Res. 29(3):573-576.

Seclaman E., Narita D., Anghel A., Cireap N., Ilina R., Sirbu I. O., Marian C. (2019). MicroRNA expression in laser micro-dissected breast cancer tissue samples - a pilot study. Pathol. Oncol. Res. 25:233-239. https://doi.org/10.1007/s12253-017-0343-y.

Shaw B. L., Battle H. I. (1957). The gross microscopic anatomy of the digestive gland of the oyster Crassostrea virginica (Gmelin). Can. J. Zool. 35:325–346.

Shi L., Liang S., Luo X., Ke C., Zhao J. (2017). Microbial community of Pacific abalone (Haliotis discus hannai) juveniles during a disease outbreak in South China. Aquac. Res. 48:1080–1088. https://doi.org/10.1111/are.12950.

Small H. J., Sturve J., Bignell J. P., Longshaw M., Lyons B. P., Hicks R., Feist S. W., Stentiford G. D.(2008). Laser-assisted microdissection: a new tool for aquatic molecular parasitology. Dis. Aquat. Org. 82:151-156.

Soletchnik P., Lambert C., Costil K. (2005). Summer mortality of Crassostrea gigas (Thunberg) in relation to environmental rearing conditions. J. Shellfish Res. 24:197–207.

Tamura K., Dudley J., Nei M., Kumar S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. https://doi.org/10.1093/molbev/msm092.

Thompson F. L., Hoste B., Thompson C. C., Huys G., Swings J. (2001). The coral bleaching Vibrio shiloi Kushmaro et al. 2001 is a later synonym of Vibrio mediterranei Pujalte and Garay 1986. Syst. Appl. Microbiol. 24:516–519.

Thompson F. L., Li Y., Gomez-Gil B., Thompson C. C., Hoste B., Vandemeulebroecke K., G. S. Rupp G. S., Pereira A., De Bem M. M., Sorgeloos P., Swings J. (2003). Vibrio neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii sp. nov., isolated from the marine aquaculture environment (bivalves, fish, rotifers and shrimps). Int. J. Syst. Evol. Microbiol. 53:245-252.

Travers M. A., Basuyaux O., Le Goic N., Huchette S., Nicolas J. L., Koken M., Paillard C. (2009). Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming. Glob. Chang. Biol. 15:1365–1376. http://dx.doi.org/10.1111/j.1365-2486.2008.01764.x.

Wei-Ming, T., Wen-Ji, L., Ming, Z., Zuo-An, Y., Shi-Lei, L., Xiang-Feng, L., Hua-Lin, L., Cheng-Dong, F., 2012. Isolation, identification and pathogenicity of Vibrio chagasii from Patinopecten yessoensis. J. Fish. China, 36, 937-943.

Weisburg W. G., Barns S. M., Pelletier D. A., Lane D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697–703. https://doi.org/10.1128/jb.173.2.697-703.1991.

Wiik-Nielsen J., Løvoll M., Fritsvold C., Kristoffersen A. B., Haugland O., Hordvik I., Aamelfot M., Jirillo E., Koppang E. O., Grove S. (2012). Characterization of myocardial lesions associated with cardiomyopathy syndrome in Atlantic salmon, Salmo salar L., using laser capture microdissection. J. Fish Dis. 35:907–916. https://doi.org/10.1111/j.1365-2761.2012.01431.x.