AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AEM.02172-06v1
73/3/981    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.
Agricola
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2007, p. 981-992, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.02172-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Coral Disease Diagnostics: What's between a Plague and a Band?{triangledown}

T. D. Ainsworth,1* E. Kramasky-Winter,2 Y. Loya,2 O. Hoegh-Guldberg,1 and M. Fine3

Centre for Marine Studies and the ARC Centre of Excellence for Coral Reef Studies, The University of Queensland, Brisbane, Queensland 4072, Australia,1 Tel Aviv University, Tel Aviv, Israel,2 Faculty of Life Sciences, Bar-Ilan University, The Interuniversity Institute for Marine Science, Eilat 88103, Israel3

Received 15 September 2006/ Accepted 29 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Coral disease is considered an important factor in the recent decline of coral reefs worldwide (29, 30, 38, 64). Reports of disease and disease-like syndromes in reef-building corals have increased substantially since first being reported in 1973 (10). This increase in the incidence of disease is due in part to a better awareness of coral health but is also linked to the increased environmental stresses affecting coral reefs (30, 31, 35, 44). Between 18 and 30 diverse coral diseases and syndromes are described worldwide (29, 64, 65) on the basis of macroscopic features. Coral disease diagnosis is primarily macroscopic, taking into account characteristics such as the extent of tissue loss, tissue color, and exposure of coral skeleton. These macroscopic disease signs then become the basis for nomenclature and diagnosis. While these characteristics allow broad descriptions of change on reefs, they are unreliable for accurate disease diagnosis and do not increase our understanding of the causes of coral disease and disease progression.

The reefs of Eilat have endured several decades of high levels of anthropogenic impact (38, 39, 66). As a result, coral disease and coral mortality have increased (39, 67), resulting in decreases in coral abundance and diversity over the past 3 decades. Increasing stress from global warming and ocean acidification has also been associated with the overall declining health of Red Sea corals (40). Al-Moghrabi (5) has reported outbreaks of black band disease (BBD) in the northern Red Sea, and Antonius and Riegl (13) have reported white syndrome on the reefs of the Sinai Peninsula, with the most abundant reef-building species (Acropora hemprichii) suffering the heaviest losses. Ben-Haim et al. (18) also reported a new disease of Pocillopora damicornis from the Gulf of Aqaba. In addition, anecdotal reports of black band disease became common in the early 1990s in the Eilat side of the Gulf of Aqaba, although no study was conducted (Y. Loya, personal communication). Recently, Barash et al. (17) reported that many of the massive corals of Eilat were affected by an infectious white plague-like disease. Despite reports and field observations of these diseases and syndromes within the region, relatively little is known about the pathology, cytology, microbial ecology, and disease processes of corals in the Gulf of Aqaba.

While there have been numerous ecological and field-based studies of corals exhibiting disease (5, 10, 11, 49, 50, 51, 54, 64), few primary pathogens have been identified to date (11, 16, 18, 24, 31, 32, 34, 36, 48, 53). Also, few studies have examined the histopathological and microbial characteristics of diseased corals. BBD is considered one of the major diseases impacting coral reefs worldwide. The first report of BBD was by Antonius in 1973 (10). Since that time, BBD has been observed to affect corals worldwide, especially in polluted environments (5, 13). The black band that is typical of this disease is composed of a mixed microbial mat that is dominated by cyanobacteria and comprises sulfur-reducing and sulfur-oxidizing bacteria as well as a number of other microorganisms. The mat overgrows coral tissues, creating a toxic environment, and tissue loss is attributed to the presence of high sulfide levels (0.8 mM) in the tissues adjacent the black band (22, 54). The distinct macroscopic signs of this disease and the growth pattern of the mat in a top-down manner across polyps and tissue structures (10, 28, 54) are used as the primary method of the disease identification. BBD has been described as one of the main diseases in the Caribbean and Florida Keys, where it has been responsible for reef decline (37). Most active during the summer months, BBD is one of the most widespread and destructive coral diseases due to its high impact on massive and framework-building corals, with rates of tissue loss of up to 2 cm per day (28, 54).

White band and white plague diseases have also been described for many regions worldwide, including the Red Sea and the Gulf of Aqaba (5, 12, 13, 14, 17, 59). Field-based surveys and visual descriptions of these diseases have shown that they typically have a lesion of white, recently exposed coral skeleton. It is believed that the white band diseases have had a major role in the community structure shift occurring in the Caribbean (15). Reefs off the coast of Florida have experienced increasing occurrence of white diseases, with patterns of disease spread suggesting a highly infectious nature (49). While pathogens have been identified for some of the white diseases (24, 46, 48, 51), others have been described as having no observable microbial community (13, 20, 21, 47). The majority of casual factors underpinning the rise in coral disease have remained elusive, particularly for many of the white diseases and white syndromes (21, 64). Richardson et al. (52) have rightly called for an integrated approach to disease diagnosis that incorporates field and laboratory studies.

In the present study we demonstrate that integrating observations on microbial diversity characteristics with specific cytological observations may be a useful tool for understanding the disease process of corals and improving the basis on which disease is diagnosed. This study also points to some significant problems associated with the simplistic use of macroscopic signs as the only means of identifying coral disease, as observed in the course of studying the reef of Eilat, Gulf of Aqaba, in the summer of 2005.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Sample collection and disease identification.
Corals were surveyed visually for signs of disease in June (early summer) 2005 at depths of 1 to 18 m on coral reefs near the Marine Biological Laboratory at Eilat in the Gulf of Aqaba. Massive and branching corals were assessed for the typical macroscopic signs of disease, including tissue loss and the apparent rapid exposure of coral skeleton, which are indicative of white diseases and white syndromes, as well as general paling from bleaching and the presence/absence of black bands. All colonies exhibiting signs of disease were photographed using a Nikon CoolPix 5000 (Nikon, Inc.) digital camera inside a Subal (Steyr, Austria) underwater housing prior to sampling. Replicate tissue and skeleton samples (n = 3) were collected from corals displaying each the observed diseases and syndromes to investigate microbial populations and histopathological features of these diseases. Of the massive (favid) coral colonies observed with diseases, six classified as having BBD and six classified as having white band disease were sampled in both early and late June 2005. For the white diseases and white syndromes of other massive corals and of branching corals, replicate samples of each type (n = 3) were collected.

Sample preservation and tissue processing.
Coral samples were fixed in 4% (wt/vol) paraformaldehyde in sterile phosphate-buffered saline (pH 7.4) (20) for 12 h prior to decalcification with 20% (wt/vol) EDTA (pH 8) (63) and standard processing for paraffin embedding. Tissues were processed through washes as follows: one wash each of 70% and 80%, two of 95%, and three of 100% ethanol for 40 min each; three xylene washes for 40 min; and then three paraffin washes under vacuum for 40 min each prior to embedding in paraffin. Serial tissue sections (4 µm) were collected onto Superfrost Plus slides (Menzel, Braunschweig, Germany) for use in fluorescence in situ hybridization (FISH) and histopathology.

Fluorescence in situ hybridization.
Oligonucleotides were Cy3 labeled by Thermo Electron Corporation Pty Ltd and used in a conventional FISH protocol (6, 7, 8, 9, 41, 42, 43, 58). Paraffin sections were dewaxed through three 5-min washes in xylene and four 5-min washes in fresh 100% ethanol. The hybridization was conducted in hybridization buffer (0.9 M NaCl, 0.01% sodium dodecyl sulfate, 0.01 M Tris-HCl, pH 7.2) for 1.5 to 2 h at 46°C and was followed by a 10- to 20-min wash in prewarmed (48°C) wash buffer (0.08 M NaCl, 0.01% sodium dodecyl sulfate, 0.01 M Tris-HCl, 0.05 M EDTA) (3). A Zeiss Meta 510 confocal scanning laser microscope (Zeiss, Germany) and spectral profile imaging via the Zeiss Image Browser software were used to visualize tissue autofluorescence and probe-conferred fluorescence. Coral tissue sections treated with the FISH protocol without the application of probe were used for spectral profiling (3). This was repeated on several tissue sections for each coral species to determine the variability in fluorescence profiles. FISH probes used within this study include a universal bacterial probe mix (EUBmix) and specific group probes for {delta}-proteobacteria (GAM42A), Cytophaga-Flavobacterium (CF319), and Vibrio sp. (MV) (Table 1). These probes were selected since the corresponding bacteria represent major bacterial groups within oceanic communities and since many Vibrio spp. are pathogenic.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Bacterial probes used in FISH

 
Histopathology.
The general tissue condition associated with the lesion and the adjacent tissues of each of the diseases was ascertained following staining using Harris's hematoxylin and eosin (with phyloxine B) (HHS32 and HT110-1-32; Sigma-Aldrich Pty Ltd). The extent of mass tissue necrosis (swelling and lysis of cells and disruption of cell structure) was recorded. In situ labeling of the 3' ends of DNA fragments was used to investigate the presence and extent of programmed cell death (1, 4, 25, 26) by use of an ApopTag in situ apoptosis detection kit as per the manufacturer's recommendations (S7101; Chemicon International, Inc.). This procedure has been shown to distinguish apoptosis from necrosis by specifically detecting DNA cleavage and chromatin condensation associated with apoptosis and confirmed by the lack of necrotic morphology. Cells were defined as apoptotic if the nuclear areas of the cells were positively labeled as indicated by red staining, as opposed to the blue hematoxylin-counterstained nonapoptotic nuclei (1).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Black band disease.
A simultaneous outbreak of black band disease and white band disease in massive corals (from the genus Favia) on the reefs of Eilat (Red Sea) was observed during June 2005. Black band diseased corals showed typical signs of the disease and were characterized by a thick black microbial mat that appeared to grow over and into underlying coral tissues (Fig. 1A1 and a1). The typical macroscale signs of white band/plague disease in Favia included an apparently clear lesion border between recently exposed bare white skeleton and tissues. The lesion lacked any observable black, mixed microbial band on the coral surface (Fig. 1B1 and b1). Newly exposed clean white coral skeleton was visible at the lesion interface and away from the disease lesion (Fig. 1b1). Based on macroscale signs, these corals would be classified/diagnosed as suffering a white disease or white plague.


Figure 1
View larger version (97K):
[in this window]
[in a new window]

  		FIG. 1. Typical (panels labeled A and a) and atypical (panels labeled B and b) black band disease of Eilat reef corals in the Red Sea. Shown are underwater photographs of infected black band diseased colonies displaying typical (A1 and a1) and atypical (B1 and b1) signs of disease. Tissue necrosis and microbial penetration and disruption of tissue layers in black band diseased colonies is evident by histopathology, using hematoxylin and eosin staining of tissue sections of typical (A2 and a2) and atypical (B2 and b2) diseased colonies, and FISH of typical (A3 and a3) and atypical (B3 and b3) diseased colonies is also shown. Scale bars, 50 µm. Abbreviations: Po, polyp; Ep, epithelium; Ga, gastroderm; Ne, necrosis; Bac, bacteria. FISH and spectral codes: blue, coral tissue; green, Symbiodinium sp.; red, bacteria. Uppercase panel labels demonstrate lower-magnification images; lowercase panel labels denote higher magnification.

 
Microbial and histopathological investigation of both BBD and white band/white plague corals identified similar distinctive cyanobacterium-dominated microbial mats. In the BBD coral, the black encircling mat was easily visible on the colony surface. In contrast, for the white disease/plague Favia coral, a similar black mat was found deep within the polyp structure and underneath the disease lesion interface. This same type of mat was identified in all sampled white band colonies in early and late June 2005 (n = 12). Given the similarity in cytological responses and microbial communities, we refer to this type of white band as atypical BBD (aBBD). Similar distinct cyanobacterium-dominated microbial communities were observed in both BBD and aBBD corals and appeared to cause tissue lysis and necrosis (compare Fig. 1A2 and a2 with Fig. 1B2 and b2). Imaging of the microbial communities identified cyanobacteria of similar morphotypes dominating the microbial communities in both the typical (Fig. 1A3 and a3) and atypical (Fig. 1B3 and b3) black band diseased colonies.


Figure 2
View larger version (97K):
[in this window]
[in a new window]

  		FIG. 2. Diseased Hydnophora sp. (A1) with evident rapid tissue loss and clear lesion borders between the coral tissue and the recently exposed skeleton (a1). Histopathology showed extensive tissue necrosis of cell layers (A2) including epithelium and gastroderm (a2). FISH revealed an extensive microbial population by use of the EUB universal bacterial probe (A3 and a3) associated with tissue necrosis and adjacent tissues. Scale bars, 50 µm. Abbreviations: Ep, epithelium; Ga, gastroderm; Zx, Symbiodinium sp.; Ne, tissue necrosis; Bac, bacteria. FISH and spectral color codes: blue, coral tissue; green, Symbiodinium sp.; red, bacteria. Uppercase panel labels demonstrate lower-magnification images; lowercase panel labels denote higher magnification.

 

Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		FIG. 3. FISH identified bacterial communities associated with diseased Hydnophora sp. identified with EUBmix (a) as belonging to {delta}-proteobacterium (b) and filamentous Cytophaga-Flavobacterium (c) groups. Populations of a Vibrio species were also identified (d). Scale bars, 50 µm. Abbreviations: Ep, epithelium; Ga, gastroderm; Zx, Symbiodinium sp.; Ne, tissue necrosis; Bac, bacteria; GAM, {delta}-proteobacteria; CF, Cytophaga-Flavobacterium; VB, Vibrio sp. FISH and spectral color codes: blue, coral tissue; green, Symbiodinium sp.; red, bacteria.

 
White disease and white syndrome.
Both massive and branching corals on the Eilat reefs appeared to be suffering from white disease, typified by abrupt lesions and areas of exposed skeleton adjacent to otherwise normally pigmented tissues. White disease was identified for the massive colonies of Hydnophora sp. (Fig. 2A1 and a1) and Porites sp. as well as for the branching coral Stylophora pistillata (see Fig. 4A1 and a1). White syndrome was identified for colonies of the plating coral Acropora sp. (see Fig. 5A1 and a1) in the region.


Figure 4
View larger version (107K):
[in this window]
[in a new window]

  		FIG. 4. White disease of Stylophora pistillata displaying tissue loss and partial bleaching in patterns starting at the branch base and progressing rapidly up the branches and through the colony (A1 and a1), with 50% mortality of the colony evident (A1). Hematoxylin and eosin staining shows that tissue structures remain intact at the lesion border (A2 and a2) with some regions of structural loss of the gastroderm adjacent to the lesions (aa2), and FISH using the EUB general bacterial probe identified large populations of bacteria associated with tissues adjacent to the lesion border (A3), a mixed bacterial population dominated by {delta}-proteobacterial taxa (a3). Scale bars, 50 µm. Abbreviations: Ep, epithelium; Ga, gastroderm; Zx, Symbiodinium sp.; Ne, tissue necrosis; Bac, bacteria; L, lesion; PB, partial bleaching. FISH and spectral color codes: blue, coral tissue; green, Symbiodinium sp.; red, bacteria. Uppercase panel labels demonstrate lower-magnification images; lowercase panel labels denote higher magnification.

 

Figure 5
View larger version (86K):
[in this window]
[in a new window]

  		FIG. 5. White syndrome of Acropora sp. (A1) showing a clear lesion boundary between the healthy tissue and the recently exposed skeleton (a1). Investigation of the tissue structure by use of hematoxylin and eosin shows that the tissues appear intact (A2) with no evidence for mass loss of tissue structure or integrity (a2). There is a distinct lack of microbial communities associated with the disease lesion (A3) and no bacterial penetration of colonization of the tissue layers (a3). Scale bars, 50 µm. Abbreviations: Ep, epithelium; Ga, gastroderm; Zx, Symbiodinium sp.; Ne, tissue necrosis; Bac, bacteria; Agg, bacterial aggregates. FISH and spectral color codes: blue, coral tissue; green, Symbiodinium sp.; red, bacteria. Uppercase panel labels demonstrate lower-magnification images; lowercase panel labels denote higher magnification.

 
There were distinct cytological and microbial differences between these macroscopically similar syndromes. The white disease of Hydnophora sp. was characterized by extensive tissue breakdown and mass tissue necrosis within the lesion areas (Fig. 2A2 and a2). The lesions were also extensively populated by bacteria, which were found to penetrate all tissue layers and infiltrate adjacent regions of tissue (Fig. 2A3 and a3). Investigation of several tissue sections showed that the bacteria were from both {delta}-proteobacterium (Fig. 3b) and Cytophaga-Flavobacterium (Fig. 3c) groups, and communities of Vibrio sp. were also found within the lesion (Fig. 3d).

In contrast, the white disease observed for the branching coral S. pistillata (Fig. 4A1) exhibited patches of bleaching close to the lesion border and regions of tissue loss (Fig. 4a1). For this white disease or form of white disease, extensive bacterial communities were found in tissue layers associated with the lesion (Fig. 3A3 and a3), and a variety of bacterial groups were evident, with no single bacterial group being dominant. There was a lack of evidence for extensive tissue breakdown and necrosis (Fig. 3A2 and a2), with only small regions of necrosis in the lesion of white disease of S. pistillata; this necrosis was limited to regions of the gastroderm (Fig.4aa2) and presumably was associated with macroscopic areas of bleaching.

Disease signs consistent with descriptions of white syndrome were also observed for the plating Acropora sp., with clear lesions evident between apparently healthy tissues and recently exposed skeleton. The white syndrome identified on Acropora sp. (Fig. 5A1) had a distinctly clear lesion border that appeared to be moving quickly across affected corals, as determined by the lack of macroscopic algal overgrowth over the large exposed areas of skeleton. The exposed skeleton at the lesion was free of any evidence for macroscopic algal overgrowth (Fig. 5a1), and tissues at the lesion border appeared healthy despite disease progression (Fig. 5B1 and b1). Microscopic analysis of the lesion border showed it to be distinctly devoid of any significant bacterial populations, with tissue adjacent to the lesion lacking any evidence of tissue breakdown or necrosis associated with the disease (Fig. 5A3 and a3). This provides clear evidence of distinct microbial community differences and morphological differences between white syndrome and white diseases (Table 2); these differences require further investigation.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Summary of microbial and disease characteristics of coral diseases in Eilata

 
Evidence of necrosis and apoptosis in disease.
Hematoxylin and eosin staining of tissue sections revealed extensive evidence for necrosis associated with the tissue loss in favid corals with black band disease. Tissue breakdown consistent with necrosis was also evident for white disease in Hydnophora sp. and Porites sp. Comparatively few or no signs of necrosis or mass loss of tissue integrity were evident for the white disease of S. pistillata or Acropora sp. In situ end labeling of fragmented DNA, used as a marker of programmed cell death, showed no evident staining in tissues of massive corals with either typical (Fig. 6a) or atypical (Fig. 6b and c) black band disease or in any of the tissues associated with the necrotic white disease of Hydnophora sp. (Fig. 6e to g). However, positive staining was evident in large regions of tissues associated with white syndrome of Acropora sp. within both epithelial (Fig. 6h) and gastrodermal (Fig. 6i) tissue layers, while no staining was evident in healthy tissue in the same colonies (Fig. 6j). Positive staining was also evident with the tissue layer samples of white disease of S. pistillata (Fig. 6k and l). However, this staining was limited to cells directly adjacent to the tissue lesions; cells away from the lesion border were not so stained (Fig. 6m). Cells showing morphology associated with necrotic cell death showed no staining when in situ end labeling for fragmented DNA was used (Fig. 6a to f). Therefore, cell death markers consistent with programmed cell death were identified only in the apparently bacterium-free lesion for Acropora sp. white syndrome and in the lesion edge for the white disease of S. pistillata and were not associated with BBD or found where mass tissue necrosis was evident (Table 2), again demonstrating that white syndrome is distinctly different from white diseases. In this case, the differentiation between white syndrome and white diseases is based on the patterns of cell death associated with the necrotic diseases (Table 2).


Figure 6
View larger version (115K):
[in this window]
[in a new window]

  		FIG. 6. In situ end labeling of fragmented DNA evident of apoptotic cell death is not detected for atypical (a) or typical (b and c) black band disease or for white diseases of Hydnophora sp. (d, e, and f). Positive staining was evident at the lesion border for the white disease of Stylophora pistillata (g and h) but not for tissues away from the border (i), and a high density of apoptotic cells was associated with white syndrome of Acropora sp. at the lesion border (k and l) but not away from the lesion border (m).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
While coral disease has been reported from the reefs of the northern Red Sea (5, 13, 14, 17, 39, 40, 59), this is the first study to explore histopathological changes and in situ microbiology associated with these diseases and syndromes. This approach to the study of coral disease has revealed that dependence on visual macroscopic characteristics alone is unreliable for accurate diagnosis and overlooks critical information on the cellular and microbial processes associated with disease-like states. The incorporation of physiological assays, for example, descriptions of photosynthetic assimilate translocation (27, 55), which demonstrate colonial activity and integration, and assays addressing host mechanisms (33) are also useful in understanding the coral colony responses to different diseases and may provide important information for disease diagnosis. We suggest that studies going beyond macroscopic disease signs by incorporating cytological, microbial, and physiological assays provide a greater level of detail for truly understanding the processes of disease in corals and will provide a better basis upon which to make accurate disease diagnosis (Fig. 7). Accurate diagnosis of coral disease can direct research and management practices towards addressing and managing the true underlying cause of disease on reefs.


Figure 7
View larger version (60K):
[in this window]
[in a new window]

  		FIG. 7. Profiling the interaction of holobiont changes and multiple levels of disease signs can be used as a basis for identifying specific criteria for disease diagnosis and to improve understanding of divergent disease processes underlying coral disease progression.

 
Atypical black band disease.
Rosenberg and Ben-Haim (59) and Barash et al. (17) recently reported an anomalous temperature spike in the Red Sea and a simultaneous outbreak of black band disease and white plague in the summer months from 2001 to 2004. Attempts by these authors to determine causative agents of the apparent white plague experienced difficulties in pathogen culturing and transmission of a suspected pathogen. In the present study, histopathological investigation revealed the presence of cyanobacterium-dominated microbial mats deep within the coral polyp structure of Favia exhibiting characteristics of white disease/plague. These microbial populations and the pattern of coral tissue disruption were consistent with those observed for typical black band disease, where cyanobacterial filaments were found penetrating tissues deep within the polyp structure. Based on the fact that the apparently white plague-infested corals all contained cyanobacterial mats deep in the tissues, this disease state appears to be an atypical form of black band disease; in addition, it appears that the black band consortium can cause an apparent white disease or that there can be a progression from a white disease into black band disease. We suggest the use of the term "atypical black band disease" to describe this different form of black band disease. This conclusion is supported by the results of Bythell et al. (21), who previously observed an apparent progression from white plague to black band disease in the Caribbean. The difficulties in pathogen identification and infection studies experienced by Barash et al. (17) may be due to the fact that some of these diseased corals were actually infected by atypical BBD, illustrating the confusion that relying on macroscopic characteristics alone may incur. We also suggest that the incidence of BBD on reefs may be underestimated if this type of atypical form is common. Further studies investigating BBD should also attempt to determine the extent and impact of atypical forms of this disease in other regions.

White diseases and white syndrome.
White diseases and white syndrome of corals showed marked differences evident from both microbiological and cytological investigation; here we define white diseases and white syndrome of corals based on specific microbial and cytological parameters. White diseases characteristically show extensive bacterial infiltration of the lesion tissue layers and tissues adjacent to the disease lesion, as well as extensive necrosis, loss of tissue structure, and/or symbiont loss. White diseases also characteristically showed little or no evidence for programmed cell death associated with the disease lesions. The term "white disease" may encompass a range of diseases or states of the disease, as seen in the example of white disease of S. pistillata in this study. A similar macroscopic pattern of tissue loss was apparent in the white disease of S. pistillata and the white disease of Hydnophora sp., yet no mass loss of tissue structure and only small regions of necrosis of the tissue layers were apparent in the white disease of S. pistillata. However, dense and mixed microbial communities were evident within the tissue layers characterizing the white diseases of both massive and branching colonies; bacteria of Cytophaga-Flavobacterium and {delta}-proteobacterial groups were present in both, as were communities of Vibrio sp., yet no single bacterial group appeared dominant. The potential of secondary colonizers involved in white diseases and the role of opportunistic pathogens in white disease progression may be very important, as evidenced by the diverse microbial community of these diseases and also considering the source of highly diverse microbial environments of the coral holobiont (19, 28, 45, 60, 61) and the coral reef.

In contrast, there was a distinct lack of microbial community interaction associated with the disease lesion of white syndrome. The disease lesion of white syndrome is characteristically free of any evidence of mass tissue breakdown; with no loss of structural integrity or necrosis, apparently healthy tissues bordered exposed coral skeleton at the disease lesion. Extensive in situ end labeling of fragmented DNA, suggestive of programmed cell death or apoptosis, was associated not only with the tissue layers of the white syndrome lesion but also with the adjacent tissues not directly associated with the disease lesion. This is consistent with previous observations of white syndrome of tabular Acropora sp. on the Great Barrier Reef (4, 56, 57). Observations of extensive programmed cell death associated with white syndrome and a lack of resource translocation to the white syndrome lesion site suggested the disease to be a host reaction progressing independent of an apparent pathogen (4, 56). We suggest that white syndrome of the corals of Eilat may be typical of a similar host reaction, or white syndrome, and that the syndrome is a disease progression divergent from that seen for necrotic and bacterial white diseases. We therefore define white syndrome of coral as a disease state that progresses independent of an apparent pathogen or microbial colonization, with little or no evidence for mass tissue necrosis and with extensive programmed cell death associated with the lesion. Previous studies of coral disease have also suggested that white syndrome is a distinct coral disease apparently linked to a lack of an observable microbial population associated with the disease progression (13, 21).

In this study, we differentiate white syndrome from white diseases based on specific cytological/morphological and microbial differences. Weil et al. (65) have defined a syndrome as a disease that has yet to have a causative agent identified. However, we suggest adhering to a medical definition of a syndrome as a collection of specific signs that occur together to characterize the particular disease or abnormality. We suggest that microbial, cytological, and physiological characteristics are useful criteria for differentiating coral diseases (Fig. 7) and for determining the specific signs suitable for accurate disease diagnosis. This also allows for the addition of other criteria that may further differentiate these diseases, as shown in Fig. 7. Furthermore, we postulate that the mechanisms of tissue loss and disease progression in white diseases and white syndrome are divergent and indicate the importance of understanding the physiological mechanisms that are associated with these diseases to truly identify the underlying cause of their increases worldwide.

Primary versus opportunistic pathogens.
Opportunistic pathogens are defined as those which infect compromised or previously stressed individuals, whereas primary pathogens are those that cause disease in an uncompromised host (62). The involvement of opportunistic as opposed to primary pathogens has been overlooked in coral disease research and needs to be considered when attempting to understand disease and determine disease causation, disease progression, and colony mortality. This is even more evident when the rapidly changing and highly impacted environment of coral reefs is considered. Increased environmental stress and pollution may destabilize the coral holobiont, creating increased potential for possible opportunistic pathogens to affect the stressed corals. The question of differentiating microbial pathogens involved in disease initiation from those involved in disease progression or sources of secondary infections is of great importance. Future work addressing disease causation should consider the definitions of the two types of pathogens: a primary pathogen is the first infecting pathogen in an uncompromised host (62), and a secondary pathogen is one that causes a secondary or subsequent infection by multiplying within already diseased tissue but that is not the primary pathogen (2). Finally, an opportunistic pathogen is defined as an organism that is normally commensal but gives rise to infection in compromised hosts (62). The use of a range of diagnostic techniques will allow us to better understand the processes of disease and address questions of the underlying causes of disease progression in dense and diverse microbial ecosystems.


   CONCLUSIONS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
This study has demonstrated the importance of cytological and in situ microbiological studies in the investigation of coral disease. Comparative analysis of disease states indicates that the current use of macroscopic disease signs is insufficient for characterizing and understanding coral disease. A particularly good example is that of black band disease within this study. The extent and impact of black band disease on reefs worldwide may be underestimated if the lack of macroscopic disease signs, as observed for Favia in Eilat, is common. Black band disease etiology and epidemiology require further analysis, especially to determine if there is the potential for other diseases to progress into this disease during summer months. Our study demonstrates that research depending solely on macroscopic signs of disease runs the risk of misdiagnosing diseases among corals.

We strongly suggest that studies incorporating cytological, microbiological, and physiological investigations provide critical insights into the inception, progression, and causal factors underpinning the current global increase in coral disease. Accurate diagnosis of coral disease is vital in providing researchers and managers with a better basis for understanding disease causation on reefs. We therefore conclude that future studies of coral disease, whether they be field or laboratory based, must examine fundamental cellular characteristics and physiological processes underlying disease progression.


   ACKNOWLEDGMENTS
 
We thank the Eilat Coral Beach Nature Reserve and David Zakai for assistance and expertise in sampling and surveying corals of the Eilat reefs. We thank the Centre for Advanced Light Microscopy at the University of Queensland for assistance with confocal microscopy and also Oded Yarden and Bill Leggat for conceptual, logistical, and editorial assistance.

We are grateful for support provided by the GEF Coral Reef Targeted Research Program (www.gefcoral.org) and the ARC Centre of Excellence for Coral Reef Studies (www.coralcoe.org.au).


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Marine Studies, University of Queensland, Brisbane 4072, Australia. Phone: 61 7 3365 3548. Fax: 61 7 3365 4755. E-mail: t.ainsworth{at}uq.edu.au. Back

{triangledown} Published ahead of print on 8 December 2006. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

  1. Adle-Biassette, H., J. E. Bell, A. Creange, V. Sazdovitch, F. J. Authier, F. Gray, J.-J. Hauw, and R. Gherardi. 1998. DNA breaks detected by in situ end labelling in dorsal root ganglia of patients with AIDS. Neuropathol. Appl. Neurobiol. 24:373-380.[CrossRef][Medline]
  2. Agrios, G. N. 1997. Plant pathology, 4th ed., Academic Press, San Diego, CA.
  3. Ainsworth, T. D., M. Fine, L. L. Blackall, and O. Hoegh-Guldberg. 2006. Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities. Appl. Environ. Microbiol. 72:3016-3020.[Abstract/Free Full Text]
  4. Ainsworth, T. D., E. C. Kvennefors, L. L. Blackall, M. Fine, and O. Hoegh-Guldberg. Disease and cell death in white syndrome of Acroporid corals on the Great Barrier Reef. Mar. Biol., in press.
  5. Al-Moghrabi, S. M. 2001. Unusual black band disease (BBD) outbreak in the northern tip of the Gulf of Aqaba (Jordan). Coral Reefs 19:330-331.[CrossRef]
  6. Amann, R., J. Snaidr, M. Wagner, W. Ludwig, and K. H. Schleifer. 1996. In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178:3496-3500.[Abstract/Free Full Text]
  7. Amann, R., B. M. Fuchs, and S. Behrens. 2001. The identification of micro-organisms by fluorescence in situ hybridisation. Curr. Opin. Biotech. 12:231-236.[CrossRef][Medline]
  8. Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919-1925.[Abstract/Free Full Text]
  9. Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.[Abstract/Free Full Text]
  10. Antonius, A. 1973. New observations on coral destruction in reefs. Abstr. Assoc. Isl. Mar. Lab. Caribb. 10:3.
  11. Antonius, A. 1977. Coral mortality in reefs: a problem for science and management, p. 617-623. Proceedings of the 3rd International Coral Reef Symposium, vol. 2. Rosenstiel School of Marine and Atmospheric Science, Miami, FL.
  12. Antonius, A. 1981. The ‘band’ diseases in coral reefs, p. 7-14. Proceedings of the 4th International Coral Reef Symposium, vol. 2. Marine Science Center, University of the Philippines, Manila, Philippines.
  13. Antonius, A., and B. Riegl. 1997. A possible link between coral diseases and a corallivorous snail (Drupella cornus) outbreak in the Red Sea. Atoll Res. Bull. 447:1-9.
  14. Antonius, A., and B. Riegl. 1998. Coral diseases and Drupella cornus invasion in the Red Sea. Coral Reefs 17:48.[CrossRef]
  15. Aronson, R. B., and W. F. Precht. 2001. White-band diseases and the changing face of Caribbean coral reefs. Hydrobiologia 460:25-38.[CrossRef]
  16. Banin, E., T. Israely, M. Fine, Y. Loya, and E. Rosenberg. 2001. Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloi to its host. FEMS Microbiol. Lett. 199:33-37.[CrossRef][Medline]
  17. Barash, Y., R. Sulam, Y. Loya, and E. Rosenberg. 2005. Bacterial strain BA-3 and a filterable factor cause a white plague-like disease in corals from the Eilat coral reef. Aquat. Microbiol. Ecol. 40:183-189.
  18. Ben-Haim, Y., and E. Rosenberg. 2002. A novel Vibrio sp pathogen of the coral Pocillopora damicornis. Mar. Biol. 141:47-55.[CrossRef]
  19. Breitbart, M., R. Bhagooli, S. Griffin, I. Johnston, and F. Rowher. 2005. Microbial communities associated with skeletal tumors on Porites compressa. FEMS Microbiol. Lett. 243:431-436.[CrossRef][Medline]
  20. Bythell, J. C., M. R. Barer, R. P. Cooney, J. R. Guest, A. G. O'Donnell, O. Pantos, and M. D. A. Le Tissier. 2002. Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals. Lett. Appl. Microbiol. 34:359-364.[CrossRef][Medline]
  21. Bythell, J. C., O. Pantos, and L. Richardson. 2004. White plague, white band and other "white" diseases, p. 351-363. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
  22. Carlton, R., and L. L. Richardson. 1995. Oxygen and sulphide dynamics in a horizontally migrating cyanobacterial mat: black band disease of corals. FEMS Microbiol. Ecol. 18:155-162.
  23. Daims, H., A. Brühl, R. Amann, K. H. Schleifer, and M. Wagner. 1999. The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22:434-444.[Medline]
  24. Denner, E. B. M., G. Smith, H. J. Busse, P. Schumann, T. Narzt, S. W. Polson, W. Lubitz, and L. L. Richardson. 2003. Aurantimonas coralicida gen. nov., sp. nov., the causative agent of white plague type II on Caribbean scleractinian corals. Int. J. Syst. Evol. Microbiol. 53:1115-1122.[Abstract/Free Full Text]
  25. Dunn, S. R., J. C. Bythell, M. D. A. Le Tissier, W. J. Burnett, and J. C. Thomason. 2002. Programmed cell death and cell necrosis activity during hyperthermic stress induced bleaching of the symbiotic sea anemone Aiptasia sp. J. Exp. Mar. Biol. Ecol. 272:29-53.[CrossRef]
  26. Dunn, S. R., J. C. Thomason, M. D. A. Le Tissier, and J. C. Bythell. 2004. Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration. Cell Death Differ. 11:1213-1222.[CrossRef][Medline]
  27. Fine, M., U. Oren, and Y. Loya. 2002. Bleaching effect on regeneration and resource translocation in the coral Oculina patagonica. Mar. Ecol. Prog. Ser. 234:119-125.
  28. Frias-Lopez, J., A. L. Zerkle, G. T. Bonheyo, and B. W. Fouke. 2002. Partitioning of bacterial communities between seawater and healthy, black band diseased, and dead coral surface. Appl. Environ. Microbiol. 68:2214-2228.[Abstract/Free Full Text]
  29. Green, E. P., and A. W. Bruckner. 2000. The significance of coral disease epizootiology for coral reef conservation. Biol. Conserv. 96:347-361.[CrossRef]
  30. Harvell, C. D., K. Kim, J. M. Burkholder, R. R. Colwell, P. R. Epstein, E. E. Grimes, et al. 1999. Emerging marine diseases—climate links and anthropogenic factors. Science 285:1505-1510.[Abstract/Free Full Text]
  31. Harvell, C. D., C. E. Mitchell, J. R. Ward, S. Altizer, A. P. Dobson, R. S. Ostfeld, and M. D. Samuel. 2002. Climate warming and disease risks for terrestrial and marine biota. Science 296:2158-2162.[Abstract/Free Full Text]
  32. Kim, K., C. D. Harvell, P. D. Kim, G. W. Smith, and S. M. Merkel. 2000. Fungal disease resistance of Caribbean sea fan corals (Gorgonia spp). Mar. Biol. 36:259-267.
  33. Kim, K., P. D. Kim, A. P. Alker, and C. D. Harvell. 2000. Chemical resistance of gorgonian corals against fungal infections. Mar. Biol. 137:393-401.[CrossRef]
  34. Kushmaro, A., Y. Loya, M. Fine, and E. Rosenberg. 1996. Bacterial infection and coral bleaching. Nature 380:396.
  35. Kushmaro, A., E. Rosenberg, M. Fine, Y. Ben-Haim, and Y. Loya. 1998. Effect of temperature on bleaching of the coral Oculina patagonica by Vibrio shiloi AK-1. Mar. Ecol. Prog. Ser. 171:131-137.
  36. Kushmaro, A., E. Banin, Y. Loya, E. Stackebrandt, and E. Rosenberg. 2001. Vibrio shiloi sp nov the causative agent of bleaching of the coral Oculina patagonica. Int. J. Syst. Evol. Microbiol. 51:1383-1388[Abstract]
  37. Kuta, K. G., and L. L. Richardson. 1996. Abundance and distribution of black band disease of corals in the northern Florida Keys. Coral Reefs 15:219-223.[CrossRef]
  38. Lesser, M. P. 2004. Experimental biology of coral reef ecosystems. J. Exp. Mar. Biol. Ecol. 300:217-252.[CrossRef]
  39. Loya, Y., and E. Kramarsky-Winter. 2003. In situ eutrophication caused by fish farms in the northern Gulf of Eilat (Aqaba) is beneficial for its reefs: a critique. Mar. Ecol. Prog. Ser. 261:299-303.
  40. Loya, Y. 2004. The coral reefs of Eilat—past, present and future: three decades of coral community structure studies, p. 1-27. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
  41. Manz, W., R. Amann, W. Ludwig, M. Wagner, and K. H. Schleifer. 1992. Phylogenetic oligonucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15:593-600.
  42. Manz, W., G. Arp, G. Schumann-Kindel, U. Szemzyke, and J. Reitner. 2000. Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridisation reveals the spatial arrangement of bacteria in sponge tissue. J. Microbiol. Methods 40:125-134.[CrossRef][Medline]
  43. Moreno, Y., C. R. Arias, H. Meier, E. Garay, and R. Aznar. 1999. In situ analysis of the bacterial communities associated to farmed eel by whole-cell hydridisation. Lett. Appl. Microbiol. 29:160-165.[CrossRef][Medline]
  44. Mullen, K. M., E. C. Peters, and C. D. Harvell. 2004. Coral resistance to disease, p. 377-394. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
  45. Pantos, O., and J. C. Bythell. 2006. Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture-independent 16S rRNA techniques. Dis. Aquat. Org. 69:79-88.[Medline]
  46. Patterson, K. L., J. W. Porter, K. B. Ritchie, S. W. Polson, E. Mueller, E. C. Peters, D. L. Santavy, and G. W. Smith. 2002. The eitiology of white pox a lethal disease of the Caribbean Elkhorn coral Acropora palmata. Proc. Natl. Acad. Sci. USA 99:8725-8730.[Abstract/Free Full Text]
  47. Peters, E. C. 1984. A survey of cellular reactions to environmental stress and disease in Caribbean scleractinian corals. Helgol. Meeresunters. 37:113-137.
  48. Peters, E. C., J. J. Oprandy, and P. P. Yevich. 1983. Possible causal agent of "white band disease" in Caribbean acroporids corals. J. Invertebr. Pathol. 41:394-396.[Medline]
  49. Porter, J. W., P. Dunstan, W. C. Jaap, K. L. Patterson, Y. Kosmynin, O. W. Meir, M. E. Patterson, and M. Parsons. 2001. Patterns of spread of coral disease in the Florida Keys. Hydrobiologia 460:1-24.[CrossRef]
  50. Richardson, L. L., W. M. Goldberg, R. G. Carlton, and J. C. Halas. 1998. Coral disease outbreak in the Florida Keys: plague type II. Rev. Biol. Trop. 46:187-198.
  51. Richardson, L. L., W. M. Goldberg, K. G. Kuta, R. B. Aronson, G. W. Smith, K. B. Ritchie, J. C. Halas, et al. 1998. Florida's mystery coral killer identified. Nature 392:557-558.[CrossRef]
  52. Richardson, L. L., G. W. Smith, K. B. Ritchie, and R. G. Carlton. 2001. Integrating microbiological microsensor molecular and physiologic techniques in the study of coral disease pathogenesis. Hydrobiologia 460:71-89.[CrossRef]
  53. Richardson, L. L., and K. G. Kuta. 2003. Ecological physiology of the black band disease cyanobacterium Phormidium corallyticum. FEMS Microbiol. Ecol. 43:287-298.[CrossRef]
  54. Richardson, L. L. 2004. Black band disease, p. 325-334. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
  55. Ritchie, K. B., and G. W. Smith. 1997. Physiological comparison of bacterial communities from various species of scleractinian corals, p. 521-526. Proceedings of the 8th International Coral Reef Symposium, vol. 1. Smithsonian Tropical Research Institute, Panama City, Panama.
  56. Roff, G., O. Hoegh-Guldberg, and M. Fine. 2006. Intra-colonial response to Acroporid "white syndrome" lesions in tabular Acropora spp. (Scleractinia). Coral Reefs 25:255-264. doi:10.1007/s00338-006-0099-4.
  57. Roff, J. 2004. Tabular Acropora syndrome on the Great Barrier Reef. Honours thesis, University of Queensland, Brisbane, Australia.
  58. Roller, C., M. Wagner, R. Amann, W. Ludwig, and K. H. Schleifer. 1994. In situ probing of gram positive bacteria with high G+C content using 23S rRNA-targeted oligonucleotides. Microbiology 140:2849-2858.[Abstract]
  59. Rosenberg, E., and Y. Ben-Haim. 2002. Microbial diseases of corals and global warming. Environ. Microbiol. 4:318-326.[CrossRef][Medline]
  60. Rowher, F., M. Breitbart, J. Jara, F. Azam, and N. Knowlton. 2001. Diversity of bacteria associated with the Caribbean coral Montastrea franksi. Coral Reefs 20:85-95.[CrossRef]
  61. Rowher, F., V. Seguritan, F. Azam, and N. Knowlton. 2002. Diversity and distribution of coral-associated bacteria. Mar. Ecol. Prog. Ser. 243:1-10.
  62. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. 1987. General microbiology. Macmillan, London, United Kingdom. [Reprint of 1971 edition.]
  63. St John, J. A., K. T. Tisay, I. W. Caras, and B. Key. 2000. Expression of EphA5 during development of the olfactory nerve pathway in rat. J. Comp. Neurol. 416:540-550.[CrossRef][Medline]
  64. Sutherland, K. P., J. W. Porter, and C. Torres. 2004. Disease and immunity in Caribbean and Indo-Pacific zooxanthellate corals. Mar. Ecol. Prog. Ser. 266:273-302.
  65. Weil, E., G. Smith, and D. L. Gil-Agudelo. 2006. Status and progress in coral reef disease research. Dis. Aquat. Org. 69:1-7.[Medline]
  66. Wielgus, J. 2003. The coral reef of Eilat (northern Red Sea) requires immediate protection. Mar. Ecol. Prog. Ser. 263:307.
  67. Winkler, R., A. Antonius, and D. A. Renegar. 2004. The skeleton eroding band disease on coral reefs of Aqaba, Red Sea. Mar. Ecol. 25:129-144.[CrossRef]

Applied and Environmental Microbiology, February 2007, p. 981-992, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.02172-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AEM.02172-06v1
73/3/981    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.
Agricola
Right arrow Articles by Ainsworth, T. D.
Right arrow Articles by Fine, M.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals