Histopathology of sea bass (Dicentrarchus labrax) experimentally infected with Mycobacterium marinum and treated with streptomycin and garlic (Allium sativum) extract

A retrospective analysis of archived tissue blocks has revealed that mycobacteriosis was apparent in Chesapeake Bay striped bass as early as 1984. Of 37 cases available from the years 1975 to 1985, 2 fish were found positive based on histopathology and genus-specific PCR. Multi-gene sequencing places the bacteria from the 2 positive cases (1984 and 1985) within the Mycobacterium tuberculosis clade with closest resemblance to the recently described fish pathogen M. pseudoshottsii. Our data confirms that mycobacteriosis is not a new disease of Chesapeake Bay striped bass and underscores the value of archived tissues in epidemiological examinations. KEY WORDS: Mycobacteriosis · Striped bass · PCR · Paraffin-embedded tissues Resale or republication not permitted without written consent of the publisherDis Aquat Org 85: [181][182][183][184][185][186] 2009 1985 were from 'service samples,' or those brought to the Oxford Laboratory by other researchers or concerned anglers and watermen for expert opinion on disease status. Tissues were predominantly preserved in formalin-based fixatives during this time period. A total of 37 cases of wild-collected striped bass were included in this retrospective study.Histopathology. Replicate 5 µm sections were taken from archived tissue blocks and stained with hemotoxylin and eosin (H&E) for initial screening. All available sections were examined with light microscopy for the presence of granulomas, inflammatory foci, or necrotic lesions associated with mycobacterial infections. Only obvious parasitic granulomas were excluded from the initial screening. All tissues containing the above pathologies were stained with ZiehlNeelsen for visualization of acid-fast bacilli (AFB) performed at 600× magnification. Samples containing clearly visible acid-fast, non-branching rods were considered histopathology positive (AFB+). Otherwise, samples were categorized as negative.Molecular detection and identification. DNA from positive cases was extracted from paraffin-embedded tissues. Briefly, microtomes were sterilized with 70% ethanol (EtOH) and new, sterile blades were used for sectioning each block. After removal of several sections from the block's surface, 3 serial 5 µm sections were cut, specific tissue sections segregated when possible, captured on a sterile wooden applicator, and transferred directly to sterile 1.5 ml micro-centrifuge tubes. Sections were deparaffinized with xylene and DNA extracted with Qiagen's DNeasy kit according to manufacturer's instructions for paraffin-embedded tissues with modification. Three ethanol (100%) rinses were performed after the use of xylene for paraffin removal, followed by complete evaporation in a vacuum centrifuge for 15 min. Elution from spin filters was performed twice to a total volume of 70 µl. Paraffinembedded splenic tissue from a single fish challenged with Mycobacterium marinum was used as an extraction control.Samples were screened for the presence of mycobacteria using a real-time PCR assay targeting a genus-specific region ...

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Historical presence (1975–1985) of mycobacteriosis in Chesapeake Bay striped bass Morone saxatilis

Jacobs¹,

Howard²,

Rhodes³

et al. 2009

“…Cultured striped bass and sea bass Dicentrarchus labrax are particularly sensitive to infections caused by Mycobacterium marinum (Hedrick et al 1987, Colorni et al 1998). This observation has been corroborated by laboratory studies that have shown striped bass to be more susceptible to infection by M. marinum than are tilapia (Wolf & Smith 1999).…”

Section: Introductionmentioning

confidence: 99%

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops × M. saxatilis and zebrafish Danio rerio

Ostland

Watral²,

Whipps³

et al. 2008

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified.Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops × M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927 T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927 T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish. KEY WORDS: Mycobacterium marinum · Hybrid striped bass · Zebrafish · Mycobacteriosis · VirulenceResale or republication not permitted without written consent of the publisher

“…Colorni et al (1998) noted that size and number of splenic granulomas regressed in sea bass Dicentrarchus labrax following experimental infection with Mycobacterium marinum, but M. marinum is infrequently isolated from Chesapeake Bay striped bass (Rhodes et al 2004, Stine et al 2010) and was not identified by PCR and subsequent sequencing from fish in this study. The number of mesenteric granulomas regressed in striped bass experimentally infected with M. shottsii (Gauthier et al 2003), the most commonly isolated species from Chesapeake Bay striped bass (Rhodes et al 2004, Stine et al 2010.…”

Section: Samples Collected In Chesapeakementioning

confidence: 53%

Low prevalence of splenic mycobacteriosis in migratory striped bass Morone saxatilis from North Carolina and Chesapeake Bay, USA

Matsche¹,

Overton²,

Jacobs³

et al. 2010

Mycobacteriosis is a chronic bacterial disease causing an ongoing epizootic in striped bass Morone saxatilis in Chesapeake Bay, USA. Prevalence of disease is high in pre-migratory fish, and multiple species of Mycobacterium spp. have been isolated. However, prevalence of mycobacteriosis in the coastal migratory population is unknown and is of concern to multiple coastal states, as disease-related mortality may impact the long-term health of the population. Histological examinations of spleens collected from fish caught by recreational anglers during the winter fishery in coastal North Carolina (2005-2006, n = 249) and during the spring fishery in Chesapeake Bay (2006, n = 120) indicated a low prevalence of mycobacteriosis (6.8% of all fish examined) in comparison to smaller, pre-migratory Chesapeake Bay fish. Genus-level PCR and subsequent sequencing of the 16-23S intergenic transcribed spacer (ITS) region revealed that all bacteria were phylogenetically related, but species is unknown. Location of survey, gender of fish, and total length of fish had no significant effect on prevalence of mycobacteriosis, parasitic granulomas, or the density of splenic granulomas (p > 0.05). These results may indicate that either granulomas resolve after Chesapeake Bay fish enter the coastal migratory population, or that there is disease-related mortality among pre-migratory Chesapeake Bay fish.