Development and applications of Ray's fluid thioglycollate media for detection and manipulation of Perkinsus spp. pathogens of marine molluscs.

Journal of Invertebrate Pathology xxx (2015) xxx–xxx

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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip

Review

Development and applications of Ray’s fluid thioglycollate media for detection and manipulation of Perkinsus spp. pathogens of marine molluscs Christopher F. Dungan a,⇑, David Bushek b a b

Maryland Department of Natural Resources, Cooperative Oxford Laboratory, Oxford, MD 21654, United States Rutgers University, Haskin Shellfish Research Laboratory, Port Norris, NJ 08349, United States

a r t i c l e

i n f o

Article history: Received 10 December 2014 Revised 8 April 2015 Accepted 5 May 2015 Available online xxxx Keywords: RFTM Perkinsosis Perkinsus sp. Mollusc diseases Fluid thioglycollate medium

a b s t r a c t During the early 1950s, Sammy M. Ray discovered that his high-salt modification of fluid thioglycollate sterility test medium caused dramatic in vitro enlargement of Perkinsus marinus (=Dermocystidium marinum) cells that coincidentally infected several experimentally cultured oyster gill tissue explants. Subsequent testing confirmed that the enlarged cells among some oyster tissues incubated in Ray’s fluid thioglycollate medium (RFTM) were those of that newly described oyster pathogen. Non-proliferative in vitro enlargement, cell wall thickening, and subsequent blue–black iodine-staining of hypertrophied trophozoites (=hypnospores = prezoosporangia) following incubation in RFTM are unique characteristics of confirmed members of the protistan genus Perkinsus. A number of in vitro assays and manipulations with RFTM have been developed for selective detection and enumeration of Perkinsus sp. cells in tissues of infected molluscs, and in environmental samples. RFTM-enlarged Perkinsus sp. cells from tissues of infected molluscs also serve as useful inocula for initiating in vitro isolate cultures, and cells of several Perkinsus spp. from both in vitro cultures and infected mollusc tissues may be induced to zoosporulate by brief incubations in RFTM. DNAs from RFTM-enlarged Perkinsus sp. cells provide useful templates for PCR amplifications, and for sequencing and other assays to differentiate and identify the detected Perkinsus species. We review the history and components of fluid thioglycollate and RFTM media, and the characteristics of numerous RFTM-based diagnostic assays that have been developed and used worldwide since 1952 for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We also review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells. Ó 2015 Elsevier Inc. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History and composition of fluid thioglycollate media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Fluid thioglycollate medium for sterility tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ray’s fluid thioglycollate media and assay history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diverse RFTM assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Solid tissue subsample assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Hemolymph subsample assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Tissue weight normalized whole- and partial-body burden RFTM assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. RFTM assays of environmental samples and excreta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantification of infections and sample metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Development of an infection intensity ranking system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Quantification of infection intensity ranks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cross-calibration of infection intensity ranking systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author at: Maryland Dept. of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, MD 21654, United States. E-mail addresses: [email protected] (C.F. Dungan), [email protected] (D. Bushek). http://dx.doi.org/10.1016/j.jip.2015.05.004 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Dungan, C.F., Bushek, D. Development and applications of Ray’s fluid thioglycollate media for detection and manipulation of Perkinsus spp. pathogens of marine molluscs. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.05.004

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C.F. Dungan, D. Bushek / Journal of Invertebrate Pathology xxx (2015) xxx–xxx

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Additional applications for RFTM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. In vitro isolate propagation and manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. In vitro manipulations with RFTM to stimulate zoosporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. PCR assays of RFTM-assayed tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future applications for RFTM methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction During 1948–1949, Perkinsus marinus (=Dermocystidium marinum = Labyrinthomyxa marina) was recognized for the first time by several independent histopathological investigations of moribund Crassostrea virginica oysters from the Gulf of Mexico (Ray, 1996), and was soon described as a prevalent and lethal oyster pathogen by several of those investigators (Mackin et al., 1950). The protozoan genus Perkinsus was erected when morphological characteristics of P. marinus zoospores indicated that the newly recognized oyster pathogen was distinct from existing taxa (Levine, 1978; Perkins, 1996). Since erection of that genus, six additional confirmed Perkinsus species, and one of uncertain affiliation (P. qugwadi, Blackbourn et al., 1998), have been described as pathogens of marine molluscs worldwide (Villalba et al., 2004; Dungan and Reece, 2006; Moss et al., 2008). In one of the research investigations that closely followed the 1950 description of P. marinus, Ray (1952a,b) developed what became a standard in vitro assay for detecting and ranking intensities of P. marinus infections in C. virginica oysters, and which uses a fluid thioglycollate nutrient medium to induce dramatic, non-proliferative enlargement of Perkinsus sp. cells. That assay and its modifications have been used during six subsequent decades to detect Perkinsus spp. infecting numerous bivalve and gastropod marine molluscs (Lester and Davis, 1981; Azevedo et al., 1990; Maeno et al., 1999; Burreson et al., 2005). Modified RFTM assays have also been used to detect and quantify Perkinsus sp. cells in environmental water samples (Ellin and Bushek, 2006), and in feces and pseudofeces of oysters and clams (Bushek et al., 2002; Park et al., 2010). RFTM incubations have been used to prepare inocula for propagation of axenic in vitro Perkinsus sp. isolate cultures (La Peyre and Faisal, 1995; Casas et al., 2002) and to induce in vitro zoosporulation by Perkinsus spp. (Perkins and Menzel, 1966; Perkins, 1996; Casas et al., 2002; Dungan and Reece, 2006). Development of the RFTM assay was a serendipitous result of careful but unsuccessful efforts to propagate P. marinus in vitro. Ray (1954a) modified a standard fluid thioglycollate medium (FTM) for sterility tests, by increasing its salt concentration to support growth and detection of marine microbes. His purpose was to test the microbial sterility of oyster gill tissue explants that he had maintained in sterile seawater as a potential substrate for in vitro propagation of the newly discovered oyster pathogen P. marinus. Those sterility tests revealed that P. marinus cells coincidentally infecting some of the tested gill explants enlarged dramatically during incubation in the modified sterility test medium. Ray’s high-salt modification of FTM was subsequently supplemented with antibacterial and antimycotic agents that narrowed the medium’s function to detection of enlarged, viable P. marinus cells (Ray, 1952b, 1966). Assays using Ray’s fluid thioglycollate medium (RFTM) detect all but one known Perkinsus species (P. qugwadi), and they have been used in both quantitative and semi-quantitative applications for disease surveys of host mollusc populations, environmental studies, and laboratory experiments.

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We review the historic development and currently expanding applications for RFTM assays. Technical specifications and methods are reviewed for diverse RFTM-based diagnostic assays that have been developed and used worldwide during six recent decades for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We evaluate and compare infection intensity ranking systems, with the aims of crosscalibrating ranking systems and standardizing terminology. Finally, we review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells. 2. History and composition of fluid thioglycollate media 2.1. Fluid thioglycollate medium for sterility tests Fluid thioglycollate medium (FTM) is a clear nutrient broth that was developed for testing the sterility of pharmaceuticals and parenteral medical materials such as sutures and injectable drugs, because it promotes the growth and detection of both aerobic and anaerobic bacteria (Brewer, 1940). It was quickly adopted as a standard sterility test medium by the Biologics Control Division of the USA National Institutes of Health during 1941 (NIH, 1955). Based on its ability to support the growth of a broad range of bacteria, FTM was also widely adopted as a sterility test medium for detection of microbial contaminants in tissue cultures and media (McGarrity, 1979; Chan and Hsiung, 1994). A useful historic advantage of FTM as a sterility test medium lay in the ability of its thioglycollate reducing agent to also neutralize the antimicrobial toxicities of organic mercury compounds such as merthiolate and thimerosal, which were widely used as preservatives in pharmaceutical and medical products. Fluid thioglycollate medium includes peptide and carbohydrate metabolites, salts, vitamins, agar, reducing agents, and an oxidation–reduction (Eh) indicator (Table 1). The L-cystine component provides sulfhydryl reducing capacity in addition to that of the sodium thioglycollate, and viscosity from a low agar concentration (0.075% w/v) inhibits gas convection between the oxidized surface and the reduced lower regions of FTM columns in tubes or vials (U.S. Pharmacopeia, 1994). With proper handling and inoculation (Difco, 1953), FTM is capable of simultaneously revealing the growth of both aerobic and anaerobic bacterial contaminants, even among samples containing mercuric or phenol preservatives (McClung, 1940; Pittman, 1946). 2.2. Ray’s fluid thioglycollate media and assay history Conclusive early confirmation of P. marinus as a lethal oyster pathogen was sought through satisfaction of Koch’s postulates by experimental initiation of dermo disease among uninfected oysters exposed to P. marinus cells that had been axenically propagated in vitro. However, early efforts to propagate P. marinus in vitro with bacteriological and mycological nutrient media failed (Prokop, 1950; Ray and Chandler, 1955; Ray, 1996). On subsequent speculation that P. marinus might proliferate in vitro as a fastidious or


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C.F. Dungan, D. Bushek / Journal of Invertebrate Pathology xxx (2015) xxx–xxx Table 1 Fluid thioglycollate sterility test medium components, concentrations, and functions. Component

Component category

Concentration (% w/v)

Function

Casitone Glucose Yeast extract Sodium chloride

Enzymatic hydrosylate of casein Carbohydrate Water-soluble fraction of autolyzed yeast cells Salt Amino acid

1.50 0.50 0.50 0.25 0.05

Peptide and amino acid metabolites Metabolite Vitamins, proteins, carbohydrates Ionic osmolite (71 mOsm kg 1) Metabolite, reducing agent

Organic thiol Non-metabolic polysaccharide Eh indicator

0.05 0.075 0.0001

Reducing agent, organic mercury neutralizer Viscosity agent Colorimetric oxidation–reduction indicator

L-cystine Sodium thioglycollate Agar Resazurin

obligate associate of living oyster tissues, efforts were initiated to develop primary oyster tissue cultures to support in vitro propagation of P. marinus (Ray, 1954a, 1996). As is still true in 2015, there were no continuous oyster tissue culture cell lines available during the 1950s. Continuous beating of their epithelial cilia indicated that many of Ray’s 1952 oyster gill tissue explants survived for extended durations in antibiotic-supplemented sterile seawater cultures (Ray, 1954a; Ray and Chandler, 1955). Sterility tests were performed to confirm the apparent axenicity of those oyster gill tissue explants, using FTM prepared with seawater to enhance its detection of potential marine microbial contaminants. Those sterility assays with high-salt FTM revealed rapid (hours) development of large and abundant spherical cells among some tested gill tissue explants, and those cells were subsequently confirmed to be hypertrophied cells of the newly discovered oyster pathogen, P. marinus (Ray, 1954a, 1996). Ray’s high-salt formulation of FTM was later supplemented with inhibitors of bacterial and fungal growth, to yield a selective medium for enlargement and detection of P. marinus cells infecting oyster tissue samples (Ray, 1952a,b, 1966). That selective, high-salt medium and its modifications (Table 2) are constitutively and functionally distinct from FTM sterility test media (Table 1) and are therefore properly distinguished as Ray’s fluid thioglycollate media (RFTM). When infected mollusc tissues are incubated in RFTM, diameters of Perkinsus sp. pathogen cells typically increase five- to thirty-fold without proliferation (Stein and Mackin, 1957a), as they transform to optically refractive hypnospores (=prezoosporangia, =hypertrophied trophozoites) with diameters of 20–300 lm (Fig. 1). Although non-proliferative enlargement in RFTM is empirically confirmed only for P. marinus, proliferation in RFTM has

never been reported for any other Perkinsus sp. RFTM-enlarged P. marinus hypnospores bear thick cell walls that are resistant to both acid and alkaline hydrolysis, and which stain blue–black with 20– 30% (v/v) Lugol’s iodine solution by a reaction that differs from the similarly colored iodine staining of starch (Ray, 1954a; Stein and Mackin, 1957b) (Fig. 2). Early experiments to identify specific RFTM components that were necessary for the non-proliferative enlargement of Perkinsus sp. cells showed that the yeast extract and glucose components were consistently necessary in combination, that the casitone component provided an inconsistent positive contribution (Ray, 1954a; Ray and Chandler, 1955), and that the ionic osmolites of the original seawater diluent could be conveniently replaced by sodium chloride alone (Ray, 1954b, 1966). P. marinus cells enlarge in RFTM at temperatures of 10–30 °C, but most rapidly at 25–30 °C (Ray, 1954a; Ray and Chandler, 1955). At optimum incubation temperatures, P. marinus hypnospores frequently reach maximum sizes in 48–96 h, and consistently do so after 7 d at 20 °C. Although the original recommended salinity for RFTM was 25‰ (714 mOsm kg 1) (Ray, 1954a), enlargement of P. marinus cells in RFTM occurs when the medium is constituted at salinities (osmolalities) of 20–50‰ (571–1429 mOsm kg 1), with ionic osmolites from either sea salts or sodium chloride (Ray, 1952b, 1954b, 1966). Enlarged hypnospores in RFTM remain suitable for microscopic analysis for several weeks, or longer in the absence of overgrowth by other microbes (Ray, 1966).

Table 2 Components and their concentrations in Ray’s fluid thioglycollate medium (RFTM) and alternative Ray’s fluid thioglycollate medium (ARFTM). Component

Casitone Glucose Yeast extract Sodium chloride L-cystine Sodium thioglycollate Agar Resazurin Sodium chloride Artificial seawater salts Lipid concentrate Chloramphenicol Penicillin–streptomycin Nystatin Final osmolality (salinity) a b

Final concentration (% w/v unless specified) RFTMa

ARFTMb

1.5 0.5 0.5 0.25 0.05

1.5 0.5 0.5 0.25 0.05

0.05 0.075 0.0001 2.0 0 0 200 lg ml 1 500 U lg ml 1 200 U ml 1 714 mOsm kg

0.05 0 0 0 1.6 42–50 mg ml 50 lg ml 1 Optional 50 U ml 1 760 mOsm kg

1

(25‰)

Ray (1966). Nickens et al. (2002), McCollough et al. (2007).

1

1

(27‰)

Fig. 1. Perkinsus sp. cells after incubation in Ray’s fluid thioglycollate medium (RFTM). Unstained, RFTM-enlarged, Perkinsus sp. hypnospores in a macerated oyster rectum tissue. Bar = 50 lm.


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Perkinsus sp. cells in molluscan fecal and pseudofecal pellets have been enumerated by RFTM assays (Bushek et al., 2002; Chintala et al., 2002; Park et al., 2010), and RFTM assay methods have also been adapted for detection and quantification of Perkinsus sp. cells suspended and dispersed in disease-endemic environmental waters (Ellin and Bushek, 2006). A relatively recent RFTM assay modification substitutes a commercial alternative fluid thioglycollate medium powder that lacks both the agar and resazurin components of the original RFTM, and which is supplemented with lipids, artificial seawater salts, and antimicrobials to produce an alternative Ray’s fluid thioglycollate medium (ARFTM, Table 2) with an osmolality of 760 mOsm kg 1 (27‰) for use in whole-body ARFTM assays and others. Specific benefits from substitution of ARFTM for RFTM include enhanced enlargement of Perkinsus sp. hypnospores, as well as expedited recovery and enumeration of enlarged hypnospores following their centrifugation or filtration from the low-viscosity, agar-free ARFTM (Nickens et al., 2002; La Peyre et al., 2003; McCollough et al., 2007). 3.1. Solid tissue subsample assays Fig. 2. Perkinsus sp. cells after incubation in Ray’s fluid thioglycollate medium (RFTM). Iodine-stained, RFTM-enlarged Perkinsus sp. hypnospores in a macerated oyster rectum tissue. Bar = 50 lm.

Although large and optically refractive RFTM-enlarged Perkinsus sp. hypnospores may be identified and enumerated microscopically without staining (Ray et al., 1953; Ray, 1954a) (Fig. 1), their unique blue–black staining characteristics and spherical shapes after treatment with Lugol’s iodine solution contrasts them dramatically against surrounding mollusc tissues that typically stain amber (Fig. 2). Following enlargement of Perkinsus sp. pathogen cells by incubation of infected mollusc tissue samples in RFTM, solid tissues are removed from the medium, macerated with clean probes or blades in a pool of 20–30% (v/v) Lugol’s iodine on a microscope slide, and coverslipped for microscopic analysis (Ray, 1966; Reece and Dungan, 2006; OIE, 2012a,b). 3. Diverse RFTM assays During more than sixty years since its initial development, RFTM has been used and modified in an expanding range of assays for detection and enumeration of Perkinsus spp. in tissues from a growing number of mollusc species, and in environmental samples. Originally developed as a diagnostic assay for dermo disease in C. virginica oysters, RFTM assays have been subsequently employed in similar diagnostic assays for detection of infections by various Perkinsus spp. in numerous marine molluscs worldwide (Villalba et al., 2004; Reece and Dungan, 2006). Like its earliest applications, current RFTM assays commonly analyze subsamples of solid mollusc tissues to detect Perkinsus sp. infections, and to estimate infection intensities semiquantitatively. Mollusc tissues used for such assays include muscle, gill, labial palp, mantle, rectum, and other visceral tissues; singly or in combinations. Quantitative, tissue weight-normalized RFTM assays enumerate enlarged Perkinsus sp. cells among weighed tissue subsamples (Choi et al., 1989; Yoshinaga et al., 2010), or among entire molluscs for sensitive whole-body assays (Bushek et al., 1994; Rodríguez and Navas, 1995; Fisher and Oliver, 1996; Almeida et al., 1999; Park and Choi, 2001; Shimokawa et al., 2010). Non-lethal diagnostic assays utilize relatively small volumes of hemolymph, collected via syringe from well-vascularized host tissues (Gauthier and Fisher, 1990; Rodríguez and Navas, 1995; McLaughlin and Faisal, 1999).

3.1.1. Inoculum tissue selection The earliest diagnostic assays for P. marinus in C. virginica used subsamples of heart, rectum, gill, and mantle tissues (Ray, 1952b). The tissues were pooled to minimize bias and maximize detection of infecting P. marinus cells, including those in dispersed, focal lesions of lightly-infected oysters. Based on consistently high P. marinus lesion frequencies and pathogen cell abundances that were observed histologically among intestine epithelia of infected C. virginica oysters, as well as empirical results from analyses of different oyster tissue incubated in RFTM, Ray (1954a) recommended the standard use of rectum tissues for RFTM assays during routine oyster population health surveys. Ray (1966) subsequently recommended testing pallial mantle tissues from areas adjacent to the labial palps, which are also frequently infected. The most sensitive RFTM assays of host tissue subsamples generally target specific host tissues for which the Perkinsus sp. of interest shows strong or consistent tropisms. For diagnoses of Perkinsus chesapeaki infections among Chesapeake Bay softshell clams Mya arenaria, for example, RFTM assays of labial palp and gill tissues were found to detect more infections than similar assays of rectum or hemolymph tissues (McLaughlin and Faisal, 1999). That finding is consistent with P. chesapeaki lesion frequencies and tissue tropisms reported from histological analyses of the same clam host (McLaughlin and Faisal, 1998; Dungan et al., 2002). Based on evidence from histological observations, gill, mantle, and visceral mass tissues have been favored for RFTM assays for Perkinsus sp. infections in the venerid clams Ruditapes decussatus and R. philippinarum (Azevedo, 1989; Navas et al., 1992; Maeno et al., 1999; Lee et al., 2001; Villalba et al., 2005). Foot muscle, mantle, and gill tissues have all been used for detection of Perkinsus sp. infections in Australian abalones (Lester and Davis, 1981; Liggins and Upston, 2010). If portals of entry for Perkinsus sp. pathogens differ from the tissues in which established infections are most common, that knowledge may pragmatically inform selection of tissue subsamples for RFTM diagnostic assays, when sensitive detections of initial infections are sought (Chintala et al., 2002). 3.1.2. Seasonal and sampling error considerations Like all diagnostic assays based on results from small tissue subsamples, RFTM assays of mollusc tissue subsamples may suffer sampling error by failing to detect pathogen cells in hosts with rare, low-intensity, or focal lesions (i.e., false negatives). As a consequence, assessments of Perkinsus sp. infection prevalence that



Fig. 3. Ray’s fluid thioglycollate medium-enlarged Perkinsus sp. hypnospores in iodine-stained oyster rectum tissue. Dark iodine staining of hypnospores is most intense at the tissue margin, and greatly reduced deeper within the macerated rectum tissue. Bar = 100 lm.

are conducted with RFTM assays during annual periods of maximum Perkinsus sp. infection intensities (maximum pathogen abundances among tissues of infected hosts), will yield the highest estimates of infection prevalence for sampled mollusc populations (Bushek et al., 1994). When regular seasonal fluctuations in Perkinsus sp. infection prevalence or intensity occur, inter-annual comparisons of RFTM assay results are only legitimate for samples collected and analyzed during the same annual seasons. Along both Atlantic and Gulf of Mexico coasts of North America, for instance, P. marinus infection measures among C. virginica oysters regularly diminish during winter and spring months, and increase during late-summer and autumn months (Ray, 1954b; Crosby and Roberts, 1990; Ragone Calvo and Burreson, 1994; Bushek et al., 1994; Andrews, 1996; Bobo et al., 1997). Similar seasonal fluctuations in Perkinsus sp. infections are reported for Crassostrea gasar oysters in northeastern Brazil (da Silva et al., 2014) and for R. philippinarum clams in Korea (Yang et al., 2012).

3.1.3. Infection intensity estimates Perkinsus sp. infection intensities have been commonly estimated by ranking relative abundances of pathogen cells detected in solid tissue subsamples analyzed by RFTM assays. Infection intensity ranks have been recorded as qualitative ranks, such as absent, light, moderate, and heavy. Numerical category ranks have also been alternatively or additionally assigned to infection intensity categories, such as 0, 1, 2, 3. Various categorical numberranking scales have been used to quantify infection intensities for individual molluscs, and to calculate measures of central tendency for sampled mollusc populations. Section 4 (below) provides a detailed review of ranking systems and sample statistics for infection intensities.

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3.1.4. Analytical artifacts Analytical errors in RFTM assays are minimized or avoided by the regular use of positive and negative control samples, and by the scrupulous handling and preparation of reagents and samples to prevent artifacts from reagent degradation, or from sample or reagent cross-contaminations. To avoid confounding results from reagent quality artifacts, periodic filtration of Lugol’s iodine solutions eliminates occasional dark-colored, angular iodine crystals that may spontaneously precipitate. The intensity and uniformity of iodine staining among RFTM-enlarged Perkinsus sp. hypnospore in macerated solid tissues may vary with penetration by iodine into macerated tissues (Fig. 3), by chemical reduction of oxidative iodine by the thioglycollate and L-cystine reducing agents of residual RFTM, or by residual alkalinity in samples that have been hydrolyzed with sodium hydroxide (NaOH) (Bushek et al., 1994, unpublished data). Rigorous cleaning of instruments between handling and maceration of separate tissue samples is essential to prevent sample cross-contamination with Perkinsus sp. hypnospores. Given the known resistance of RFTM-enlarged Perkinsus sp. hypnospores to alkaline and acid hydrolysis, scrubbing instrument work surfaces briefly in a beaker containing sand and 1% (w/v) sodium hypochlorite (20% v/v commercial bleach) to hydrolyze adherent matter, rinsing in running water, and immersion in 100% ethanol before flaming, should destroy any sample residues, including DNAs (Reece and Dungan, 2006). Likewise, careful avoidance of physical contact between iodine reagent droppers and Perkinsus sp.-contaminated sample tissues or surfaces is essential to prevent transfers of Perkinsus sp. hypnospores to either the iodine reagent reservoir, or directly between tissue samples (Bushek et al., 1994). Due to the potent capabilities for PCR-amplification of minor contaminant DNAs, results from PCR assays performed on DNAs extracted from tissues previously analyzed by RFTM assays will be seriously compromised if such tissue samples were collected or manipulated in any of the many common ways that allow inadvertent contamination of analyzed tissue samples with extraneous DNAs (Bushek et al., 1994). Analytical accuracy with RFTM assays also benefits from experience in microscopically differentiating the morphological and staining characteristics of enlarged Perkinsus sp. cells from those of endogenous pigmented bodies and cells that may occur in tested mollusc tissues; as well as the accurate differentiation of non-Perkinsus sp. cells and objects that may be associated with such tissue samples. Imposters may include dark-staining pollen grains of various shapes and sizes that may have been ingested or are otherwise associated with assayed mollusc tissues. Spheroidal pollen grains may show angular faces or multiple lobes that distinguish them, and may also stain brown with Lugol’s iodine. Spherical labyrinthulid protists that are common associates of marine molluscs may proliferate in RFTM (Fisher and Oliver, 1996), but stain brown or golden with iodine. Spherical, refractive, chorioned-eggs of analyzed mollusc hosts may be numerous among assayed tissues, especially in samples collected during reproductive seasons. Endogenous, dark-pigmented host or other cells may also deceive or mislead, including ceroid cells of oysters and the pigmented symbiotic zooxanthellae of some tridacnid clams. 3.2. Hemolymph subsample assays 3.2.1. Sample tissues Analyses of mollusc hemolymph samples by RFTM assays are performed to detect systemic Perkinsus sp. infections (Gauthier and Fisher, 1990; Bushek et al., 1994; Rodríguez and Navas, 1995; Nickens et al., 2002). For bivalve molluscs, such assays often utilize non-lethal hemolymph subsamples drawn from an


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adductor muscle or other hemolymph sinus via a syringe access port notched or drilled between the valve margins, or by collecting hemolymph samples without using a mechanical access port where possible. Pericardial hemolymph can also be sampled, but with greater risk to the host mollusc. 3.2.2. Sensitivity and unique attributes Detection of Perkinsus sp. infections from mollusc hemolymph samples requires the presence of pathogen cells in systemic hemolymph circulation. Although systemic infections by Perkinsus spp. may occur among molluscs with initial or low-intensity infections, systemic lesions are more common among oysters with advanced or heavy infections (Mackin, 1951). Among oysters harboring P. marinus infections of relatively high intensities, RFTM assays of hemolymph were found by one investigation to be more sensitive and precise than those that analyzed solid mantle tissues from the same oysters (Gauthier and Fisher, 1990). However, due to their similarly low sensitivity for detection of low-intensity infections, the diagnostic sensitivities of RFTM assays using either hemolymph samples or solid tissue subsamples were lower than the sensitivity of nearly whole-oyster RFTM assays, when the three assays were performed on tissues from the same oysters (Bushek et al., 1994). Despite the reduced diagnostic sensitivity of RFTM assays performed on small samples of mollusc hemolymph, the non-lethal acquisition of diagnostic hemolymph samples enables conservative periodic re-testing of experimental host molluscs for evaluations on pathogenesis of Perkinsus sp. infections over time (Ford, 1986). 3.2.3. Infection intensity estimation The reduced density of microscopically obstructive cell debris in hemolymph samples compared to solid tissue biopsies, facilitates accurate enumeration of Perkinsus sp. hypnospores in RFTM assays of hemolymph. Sodium hydroxide lysis of hemocytes and other debris in RFTM-incubated mollusc hemolymph samples clarifies them for microscopic analyses of residual Perkinsus sp. cells in wells of multi-well plates, or captured on filter membranes (Gauthier and Fisher, 1990; Bushek et al., 1994; Nickens et al., 2002). Plating of countable quantitative dilutions of Perkinsus sp. hypnospore pellets allows accurate quantification of actual abundances of Perkinsus sp. cells, for estimation of relative infection intensities from analyses of hemolymph samples of known volumes. Results of several correlation analyses provide guidance for converting volumetric counts of P. marinus cells from RFTM assays of oyster hemolymph samples to the interval ranks of the Mackin infection intensity scale for solid tissue subsamples, and to tissue weight-normalized P. marinus burdens for whole oysters (Gauthier and Fisher, 1990; Bushek et al., 1994; Nickens et al., 2002). Similar analyses may prove valuable for other Perkinsus spp. or hosts. 3.2.4. Sources of analytical error and artifacts All of the sources for analytical errors that are previously listed here and elsewhere for solid tissue subsample RFTM assays, may also compromise RFTM assays of mollusc hemolymph samples (Bushek et al., 1994). Since hemolymph-bearing organs and tissues are typically hidden from view by the shells of bivalves and other molluscs, hemolymph samples are also characteristically drawn blind. Even with high dexterity and intimate anatomical knowledge by samplers, the qualitative integrity of hemolymph samples is inherently uncertain in most cases. Such hemolymph samples are particularly prone to include unknown proportions of pallial fluid (shell liquor), which may confound confident quantitative interpretations of assay results.

Fig. 4. Perkinsus sp. hypnospores from a whole body assays of a C. virginica oyster with Ray’s fluid thioglycollate medium, following alkaline hydrolysis of oyster tissues and staining with Lugol’s iodine. Small, insoluble non-Perkinsus sp. particles stain amber. Bar = 50 lm.

3.3. Tissue weight normalized whole- and partial-body burden RFTM assays 3.3.1. Tissues Whole tissues or tissue samples from assayed molluscs are wet-weighed, and may be macerated or homogenized before inoculation into 10–20 volumes of RFTM (Bushek et al., 1994; Fisher and Oliver, 1996) or ARFTM media (La Peyre et al., 2003; McCollough et al., 2007). Where separate tissue samples are needed for independent histological, biochemical, or DNA-based assays on RFTM-assayed molluscs (Balseiro et al., 2010), such samples may be excised and preserved before wet weights of RFTM-assayed tissues are recorded. 3.3.2. Procedure Assayed tissues are weighed in order to normalize counted Perkinsus sp. cell abundances (infection intensities) to sample tissue wet weights. Whole oyster tissues (Bushek et al., 1994), entire oyster tissue homogenates (Fisher and Oliver, 1996; McCollough et al., 2007), samples of excised individual tissues (Choi et al., 1989; Oliver et al., 1998; Chintala et al., 2002), and oyster tissue homogenate subsamples (La Peyre et al., 2003) have been incubated in RFTM or ARFTM media for 7–14 d at 20–25 °C to enlarge Perkinsus sp. cells, and to strengthen their cell walls for resistance to alkaline hydrolysis. Following RFTM incubation, host mollusc tissues are hydrolyzed for 1–6 h at 60 °C in 2 N NaOH (Choi et al., 1989; Bushek et al., 1994). Pellets of residual Perkinsus sp. hypnospores are washed by centrifugation to neutralize their pH, resuspended in a PBS-BSA buffer that inhibits their aggregation and surface adsorption (La Peyre et al., 2003), stained with 20– 30% (v/v) Lugol’s iodine, and enumerated after quantitative dilution and immobilization on microscope slides (Bushek et al., 1994), on filter membranes (Fisher and Oliver, 1996), or in microplate wells (La Peyre et al., 2003; McCollough et al., 2007) (Fig. 4).



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Counts of Perkinsus sp. hypnospores are corrected for quantified sample dilution factors, and infection intensities are reported as numbers of Perkinsus sp. cells per gram of analyzed host tissue wet weight.

incubation in RFTM. Perkinsus sp. cells excreted in feces and pseudofeces of experimentally challenged oysters were also enumerated following enlargement in RFTM, iodine staining, and immobilization on filter membranes for counting (Bushek et al., 2002).

3.3.3. Sensitivity and quantitative infection intensity estimates Whole-body RFTM assays with a theoretical sensitivity of one Perkinsus sp. cell host 1, are recommended for detection of initial infections (Ragone Calvo et al., 2003; McCollough et al., 2007) and other potential low-intensity or focal infections (Fisher and Oliver, 1996; Almeida et al., 1999). For sensitive certification assays to detect Perkinsus sp.-infected molluscs in lots proposed for introduction into regulatory jurisdictions, whole-mollusc RFTM assays are recommended on samples with statistically adequate numbers of individuals (Bushek et al., 1994; OIE, 2012a,b). Whole- or partial-body RFTM assays may also serve as fundamental tools for investigations of host/pathogen interactions where precise, quantitative measures of infection intensities among experimental oysters may reveal thresholds for lethal infection intensities (Albright et al., 2007), assess virulence differences among Perkinsus sp. pathogens and strains (Chintala et al., 2002), or identify resistant phenotypes and genotypes among mollusc hosts (Bushek and Allen, 1996). Results of several correlation analyses provide generally consistent guidance for approximating tissue weight-normalized P. marinus burdens from whole-oyster RFTM assays to the interval ranks of the Mackin infection intensity scale for solid tissue subsamples (Choi et al., 1989; Gauthier and Fisher, 1990; Bushek et al., 1994; Nickens et al., 2002).

3.4.1. Procedure Environmental water samples (500 ml) are pre-filtered through 25-lm nylon mesh filters to remove large particles, and small particles in the resulting filtrates are then collected by filtration onto a 0.45-lm cellulose acetate membrane. Perkinsus sp. cells immobilized on filter membranes are enlarged by incubation of membranes in RFTM for 7 d at 20 °C, followed by alkaline hydrolysis of the filter membranes and labile filtrate particles. Suspensions of residual Perkinsus sp. hypnospores are concentrated and washed by centrifugation in water, stained with Lugol’s iodine solution, quantitatively diluted if necessary, and counted microscopically (Ellin and Bushek, 2006). Fecal and pseudofecal samples are collected where deposited by confined experimental mollusc hosts, sedimented in centrifuge tubes, covered with 5–10 ml of RFTM, incubated at 20 °C for 7– 14 d, washed by centrifugation in deionized water, stained with Lugol’s iodine solution, quantitatively diluted if necessary, and immobilized on filter membranes for counting (Bushek et al., 2002).

3.3.4. Sources of analytical error and artifacts Alkaline lysis of samples during whole-body RFTM assays eliminates most, if not all, potentially confusing non-Perkinsus sp. cells within samples. However, cross-contamination and other sources for analytical errors may corrupt results of whole-mollusc RFTM assays. Whole-mollusc RFTM assays are theoretically free from inaccurate results due to sampling error, since all tissues of potential hosts are exhaustively analyzed (Bushek et al., 1994). Because alkaline pH inhibits iodine staining of Perkinsus sp. hypnospores, thorough neutralization of any residual NaOH lysis reagent is necessary for diagnostic iodine staining of Perkinsus sp. hypnospores (Bushek, unpubl.). If microscope counting chambers are used for enumeration of suspended Perkinsus sp. hypnospores, those should have counting chamber dimensions that are all at least twice the diameter of the largest Perkinsus sp. cell, in order to avoid physical exclusion of larger cells from the counted sample. Hemacytometers with 100-lm chamber depths commonly exclude larger Perkinsus sp. hypnospores. 3.4. RFTM assays of environmental samples and excreta RFTM assay methods have been adapted for detection and quantification of viable Perkinsus sp. cells that are suspended and dispersed in environmental waters (Ellin and Bushek, 2006). Contrary to an earlier report that Perkinsus sp. cells from environmental water samples failed to enlarge in RFTM (Yarnall et al., 2000), Ellin and Bushek (2006) estimated similar densities of P. marinus cells in environmental water samples by direct enumeration of membrane-captured pathogen cells with genus-Perkinsus immunoassays, and by RFTM assays of enlarged hypnospores on duplicate water filtration membranes. Almeida et al. (1999) also found planktonic cells that responded positively to RFTM incubation, but they did not confirm the identities of those cells despite their caution that enlarged non-Perkinsus sp. cells may have yielded false-positive results in their assays. To date, only Perkinsus spp. have been shown to respond characteristically to

3.4.2. Sensitivity, specificity, and unique attributes Assays yield abundance estimates for Perkinsus sp. cells in analyzed volumes of environmental water samples, providing a technically simple method for analyses supporting investigations of waterborne transmission dynamics by infectious Perkinsus sp. cells. Since cells of all confirmed Perkinsus species enlarge similarly in RFTM, differential estimates on abundances of individual Perkinsus species are not practical by this method for samples from several regions where multiple Perkinsus species are sympatric (Dungan and Reece, 2006; Reece et al., 2008; Arzul et al., 2012; da Silva et al., 2014). The same sources of analytical errors that are described for other RFTM assays may also confound results of RFTM assays of marine and estuarine water samples, and of excreta from infected host molluscs. 4. Quantification of infections and sample metrics As described above, RFTM assays provide information on the number of parasites present in tested tissues, which provides a measure of the degree or intensity of the infection. Since Perkinsus sp. cells are often present at densities that make actual counting impractical, relative intensities of Perkinsus sp. infections are routinely recorded and rated along ordinal scales representing increasing pathogen cell abundance in tissue biopsies (Ray, 1954b; Choi et al., 1989; Bushek et al., 1994). Means of infection intensity ranks are often exclusively reported for samples, without assay results for individuals. Unfortunately, valuable epizootiological information may be lost when only summary statistics are considered. Furthermore, current and historic uses of a wide variety of ranking systems makes cross-study comparisons difficult and potentially erroneous. In this section we review the development of infection intensity ranking systems and related statistics. 4.1. Development of an infection intensity ranking system The earliest investigations of P. marinus lesions in histological sections of C. virginica (Mackin, 1955), and subsequently in RFTM assays (Ray et al., 1953), ranked infection intensities in ordinal categories of light, moderate, and heavy, based on the relative


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Fig. 5. From Andrews and Hewatt, 1957 (Ecol. Monogr. 27, 1–25). The seasonal occurrence and intensity of Perkinsus marinus infections in moribund oyster gapers during 1953 and 1954. Each symbol represents one gaper. The number of oysters at the beginning of each warm season is shown at the top of the figure. Gapers were grouped by 2day intervals, although they were collected daily.

abundance of parasites present. As investigators gained experience, intermediate categories of light-to-moderate and moderate-toheavy were added (Ray, 1954b). Initially, assay results were summarized as frequency distributions showing the overall prevalence of infections by category, often as stacked bar plots. Such frequency distributions display the status of infections in the sample, but quickly become cumbersome as the number of samples and comparisons increases (Fig. 5). To simplify comparisons, Mackin (unpublished, cited in Ray, 1954b) assigned integer values to the ordinal

infection categories and created a summary statistic called the weighted incidence, which he calculated by weighting each assay by its infection intensity rank, summing the ranks; then dividing by the number of individuals assayed (Ray, 1954b). A category of ‘very light’ was subsequently added to represent samples with less than ten parasites, and was assigned the value of 0.5 since it was less intense than a light infection that already held the assigned value of 1; but not uninfected, which held the value of zero (Ray, personal communication).



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Fig. 6. Histograms of 4 hypothetical oyster populations with different prevalences of Perkinsus marinus infections, equivalent means for WP (mean of all individuals assayed including zeros), and mean intensities (only infected individuals) that run counter to prevalence. (A) 20% prevalence, WP = 1.0, mean intensity = 5.0. (B) 50% prevalence, WP = 1.0, mean intensity = 2.0. (C) 80% prevalence, WP = 1.0, mean intensity = 1.2. (D) 100% prevalence, WP = 1.0, mean intensity = 1.0.

Mackin, Ray, and others incorrectly reported prevalence as incidence, and also weighted incidence (WI) rather than weighted prevalence (WP) (Ragone Calvo and Burreson, 1994). Incidence measures the total number of new infections in a population of known size over a period of time, as a rate; while prevalence measures the proportion of infected individuals in a sample from a population (Margolis et al., 1982; Bush et al., 1997). The historic infection intensity ranking scale for RFTM assays is commonly known as the Mackin scale, and the sample mean for all infection ranks, including zeros, is widely termed a weighted prevalence (WP) in studies of P. marinus infections of C. virginica oysters, and sometimes for other Perkinsus spp. in other hosts. While the Mackin scale is quite useful for ranking infection intensities, it is not perfect and there are problems with the interpretation of the sample statistic WP. Calculating WP is mathematically equivalent to calculating an average intensity rank for all tested individuals, including those without detected infections that are assigned the rank of zero. However, the Mackin scale was created as a simple ordinal scale sensu Stevens (1946), who argued that calculating the mean of ordinal data is statistically inappropriate because there is no knowledge of the size or uniformity of the intervals, and their underlying distribution is typically unknown. Debate on the statistical value of averaging and analyzing ordinal data with parametric statistics continues, and many argue there is no basis for limitations on calculating such means (Velleman and Wilkinson, 1993). Regardless, calculation of WP (or any average) suffers from the fact that multiple data distributions can yield the same sample mean, with maximum variability at intermediate values. For example, Fig. 6 shows four hypothetical oyster populations with varying infection intensity distributions and prevalences of 25%, 50%, 80%, and 100%. All have identical sample means of 1.0 for infection intensities that include zeros (WP), but their epizootiological circumstances and their projected incipient mortalities vary widely. Calculating the mean infection intensity of only the infected individuals highlights this difference, providing mean intensities of 5.0, 2.0, 1.2, and 1.0, a trend opposite to that for prevalences in this example. The patterns of these disparate distributions are masked when sample data are condensed into a

single metric like WP, the mean infection intensity of all sample members. Mackin (1962) reported that oyster mortalities could be expected to increase as weighted prevalence exceeded 1.0, and this was clearly evident in data presented by Bushek et al. (2012). But because multiple distributions can lead to a given WP (e.g., Fig. 6), not all populations with that WP will experience the same mortality. Fig. 7 shows the distribution of infections from 160 samples of Delaware Bay oysters collected between 1990 and 2006, in which each sample contained both gapers (moribund) and live oysters. Samples are predominantly from the months of July through November when infections were intensifying. Oysters may die from many causes, and many oysters of Fig. 7 probably died from causes other than P. marinus infections. Note that the fractions of individuals with infection intensities below 4 were more or less equally distributed in both the live and dead groups. That is, an oyster with an infection intensity of 0.5, 1, 2, or 3 was no more likely to be dead than an oyster without any detectible infection. However, the fraction of moribund oysters nearly doubled for oysters with infection intensities of 4, and increases even further at the intensity rank of 5. We interpret this to mean that the probability of oyster death is not increased by dermo disease until infection intensities increase beyond the rank of 3 on the Mackin scale. As described above, this information is lost when infection intensities are condensed into a single sample statistic like WP. The significance of this was depicted by Bushek et al. (2012; Fig. 3) where increasing WP showed little mortality effect below the value of 1.5; but that mortalities increased dramatically at higher values of WP, with a concomitant increase in variability. That is, a population’s average infection intensity (WP), may be associated with a range of mortality rates across a factor of two. Reporting prevalences of higher-intensity infections provides an alternative statistic for projecting mortalities (Gray et al., 2009), in which it is critical to understand what infection intensity threshold increases the probability of mortality. In Fig. 6, most oysters are unlikely to experience incipient mortality from P. marinus infections; in fact, the highest mortality may be most likely in the hypothetical population with the lowest actual prevalence. Several


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directly, or normalized by sample tissue wet weights for comparison. Using this property, Choi et al. (1989) demonstrated that the Mackin scale was essentially a log10 scale of weight-normalized infection intensities (Fig. 8). Gauthier and Fisher (1990) performed a similar analysis on hemolymph samples, using sample volume instead of weight. Subsequently, Bushek et al. (1994) and Oliver et al. (1998) conducted similar analyses to compare total body burdens with hemolymph and various other tissue samples, and Rodríguez and Navas (1995) made similar comparisons with two clam species infected by Perkinsus olseni. All of these analyses demonstrated the log10 basis of the Mackin scale, meaning that the Mackin scale is effectively a quantitative, log-based interval scale. Choi et al. (1989) also demonstrated that the ranked intensity scale was continuous and not categorical, i.e., the scale could be refined to include many intermediate rankings, or measurements, should the observer wish to make such estimates. Some authors have applied standard parametric analyses to ranked intensities (e.g., Crosby and Roberts, 1990). While some may argue that such analyses violate assumptions of normality, others argue that measurements of scale should not be so restrictive in proscribing analyses (Velleman and Wilkinson, 1993). We conclude that WP is in fact an arithmetic mean of interval ranked infection intensities that corresponds to log10 transformations of pathogen cell counts. As such, calculating means and variances is appropriate. Error terms sensu Crosby and Roberts (1990) should be reported, and parametric comparisons of WP data can be made. Following the advent of the RFTM body burden protocol, several researchers abandoned the ranking of infection intensities in favor of estimating actual parasite densities (Almeida et al., 1999; Park and Choi, 2001; Choi et al., 2002; Leethochavalit et al., 2004). When the number of parasites present in a host can be determined, as is the case with the body burden protocol of Choi et al. (1989), Margolis et al. (1982), with additional input by Bush et al. (1997), define and recommend a standard terminology. Specifically, the mean intensity is the average number of parasites among only the infected individuals in a sample. This is differentiated from mean abundance, which is the average number of parasites among all individuals in a sample, including those where no parasites were detected. Although ranks from various intensity scales like the Mackin scale are not counts, mean abundance is mathematically analogous to WP, the difference being that the former uses absolute counts whereas the latter uses ordinal infection intensity ranks. When calculating means of count data that span orders of magnitude, it is important to either use log10-transformed data, or to calculate geometric means that

Fig. 7. Comparison of Mackin-scale Perkinsus marinus infection intensities in live and moribund oysters from Delaware Bay, USA. (A) Live oysters, n = 2709. (B) Moribund oysters, n = 537. (C) Probability of being dead, n = 3246.

investigators, including ourselves, separately calculate sample prevalences of infections at or above projected lethal intensities, in order to project probable imminent mortalities from perkinsosis; but such statistics are rarely published. 4.2. Quantification of infection intensity ranks A major advance in quantifying Perkinsus sp. infections occurred when Choi et al. (1989) calibrated the Mackin scale to actual pathogen abundances in order to study the energetic demands of P. marinus on its host. Choi discovered that the cell walls of enlarged hypnospores that formed in RFTM were resistant to NaOH digestion, allowing the actual parasite load of the sample to be determined. Infections among individuals could be compared

Fig. 8. From Choi et al., 1989 (J. Shellfish Res. 8, 125–131). Regression of Perkinsus marinus hypnospores g 1 oyster tissue as enumerated following alkaline hydrolysis of oyster tissues incubated in Ray’s fluid thioglycollate medium, vs. Mackin scale infection intensity category ranks assigned to the same oyster tissues by traditional microscopic infection intensity ranking.


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prevent rare heavily- or lightly-infected individuals from biasing the sample statistic (Bushek et al., 1994). Many studies do not specify that data were log-transformed for analysis, or that geometric means were calculated; so it is assumed that arithmetic means were calculated with corresponding errors. Caution should be exercised when interpreting arithmetic means of count data, and in any analyses where data span orders of magnitude. The term weighted prevalence (WP) is common, though not universal, to the lexicon of dermo disease in C. virginica, but not necessarily to forms of perkinsosis caused by other Perkinsus spp. in other hosts. Some authors have used the simple term, ‘‘mean infection intensity’’ for the arithmetic mean of the numeric interval ranks of Perkinsus sp. infection intensities estimated for all molluscs in a sample (Crosby and Roberts, 1990; Dungan and Reece, 2006; Albright et al., 2007). Others have defined terms and reported results for infection intensity means that are calculated only from values for the infected oysters in samples, such as ‘infection intensity index’ (Dungan et al., 2002), ‘mean infected body burden’ (McCollough et al., 2007), and ‘mean infection intensity’ (da Silva et al., 2014). Researchers should beware and seek clarity regarding what has been reported in any particular study, and more importantly, should take care to properly report their calculations of summary statistics and the specifics of the scales they use. When only summary statistics are reported, supplementary data that include individual intensity scores should be made available through archival data. For mollusc populations with high prevalences of Perkinsus sp. infections, mean pathogen abundances (=WP) (Bush et al., 1997) calculated for samples from such populations provide accurate general estimates of the severities of infections in those populations, and have been used to accurately predict disease mortality levels (Bushek et al., 2012). For samples harboring low prevalences or low intensities of Perkinsus sp. infections, mortality impacts projected from mean pathogen abundances will be weakened by the multiple underlying epizootiologies of such means (Fig. 6). Therefore, separate considerations of the prevalence and the mean intensity of infections among the infected segment of sampled populations provides a more reliable assessment of probable

Table 3 Some numerical Perkinsus sp. infection intensity ranking systems used to score results of Ray’s fluid thioglycollate medium assays, historically and worldwide. Number Rank values of ranks

Use location

Use species

Reference

C. virginica oysters 3 clam species Crassostrea spp. oysters

Ray et al. (1953)

4

0, 1, 3, 5

4

0, 1, 2, 3

Gulf of Mexico, USA New Zealand

5

0, 1, 2, 3, 4

Brazil

6

0, 1, 2, 3, 4, 5

7

0, 0.5, 1, 2, 3, 4, 5

Maryland, USA USA

7

0, 1, 2, 3, 4, 5, 6

8

0, 1, 2, 3, 4, 5, 6, 7

11

0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5

16

0, 0.33, 0.67. 1.33, 1.67, 2, 2.67, 3, 3.33, 4, 4.33, 4.67,

1.0, 2.33, 3.67, 5

Hine and Diggles (2002) Sabry et al. (2009), da Silva et al. (2014) Dungan et al. (2002) Ray (1954b)

2 clam species C. virginica oysters Florida, USA C. virginica Quick (1972) oysters Maryland, Oysters and Calvo et al. USA clams (1996), McLaughlin and Faisal (1999) New Jersey & Oysters and Bushek et al. clams (1994), White South et al. (1998) Carolina, USA Gulf of C. virginica Craig et al. Mexico, USA oysters (1989)

epizootiological effects (Bush et al., 1997). The products of mean infection intensity and infection prevalence yield mean abundance (or WP) (Margolis et al., 1982; Bush et al., 1997), thus the statistics prevalence, mean infection intensity, and mean abundance (or WP) can be calculated from each other as follows, where prevalence is expressed as a rational proportion rather than a percentage: Prevalence x mean infection intensity = mean abundance (or WP) Mean abundance (or WP)/mean infection intensity = prevalence Mean abundance (or WP)/prevalence = mean infection intensity. When prevalence is 100%, then mean infection intensity = mean abundance (or WP). Where possible, providing the actual frequency distribution of infection intensities will be the most informative, by revealing bimodality, skewness, or kurtosis in the data.

4.3. Cross-calibration of infection intensity ranking systems Many infection intensity ranking systems have been used historically and worldwide to score RFTM assay results (Table 3). In addition to the 7-category Mackin scale broadly used with USA oysters and others (Ray, 1954b; Balseiro et al., 2010), there are other scales that alter the range of values or the resolution of the scale or both. For example, Andrews and Hewatt (1957) as well as Ray et al. (1953) decreased the resolution of the Mackin scale by compressing all ratings into the ranks of 0, 1, 3, and 5. In contrast, Choi et al. (1989) and Craig et al. (1989) increased resolution by dividing each integer rank into thirds and eliminating the 0.5 rank; whereas Bushek et al. (1994) used half-rank ratings throughout the Mackin scale. Quick (1972) changed the bounds of the Mackin scale by reassigning values to the categories with a strict integer scale that changed the value of the lowest category from 0.5 to 1, and successively increased the values of higher infection intensity categories for a scale that ranges from 0 to 6. Several studies have used Quick’s scale; also called ‘Quick and Mackin’ (e.g., Crosby and Roberts, 1990; Bobo et al., 1997) and this scale may provide a better correspondence to body burden counts than the Mackin scale used by Choi et al. (1989) or Bushek et al. (1994). A unique 0–7 integer scale was created by Austin Farley (unpublished), but has not been widely adopted (Calvo et al., 1996). While the Mackin scale has been applied to infections by Perkinsus species in several hosts (e.g., Rodríguez and Navas, 1995), several investigators have defined different scales for uses with diverse hosts and Perkinsus species (Table 3). A 4-rank (0–3) integer scale has been used with New Zealand molluscs (Hine and Diggles, 2002), while a 5-rank (0–4) integer scale has been used with Brazilian oysters (Sabry et al., 2009; da Silva et al., 2014). Dungan et al. (2002) defined a 6-rank (0–5) integer scale they used with Maryland, USA clams. Rodríguez and Navas (1995) provide a thorough evaluation quantifying the Mackin scale

Table 4 Proposed cross-calibration of four scales for numeric interval rank categories of Perkinsus marinus infection intensities estimated by assays of Crassostrea virginica tissues with Ray’s fluid thioglycollate medium. Ranking scale (reference)

Numeric interval ranks for Perkinsus sp. infection intensities

Mackin (Ray, 1954a) Quick (1972) Farley (Calvo et al., 1996) Craig et al. (1989)

0 0 0 0

0.5 1 1 0.33

1 2 2 0.67 1.0 1.33

2 3 3 1.67 2.0 2.33

3 4 4 2.67 3.0 3.33

4 5 5 3.67 4.0 4.33

5 6 6&7 4.67 5.0


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as they applied it to Perkinsus olseni (=P. atlanticus) infections in Ruditapes spp. clams in Europe; demonstrating a very similar relationships to that found by Choi et al. (1989), Gauthier and Fisher (1990), and Bushek et al. (1994). To our knowledge, only the Mackin scale has been calibrated to pathogen tissue abundances. The application of disparate ranking systems may preclude direct comparisons among studies and data sets. As indicated above, some studies have only changed the resolution of the scale, and these studies may be compared directly, as long as it is recognized that the level of precision can never exceed the resolution of the measurement scale used. A more significant issue arises when comparing studies that use different scales altogether. Nevertheless, patterns such as the timing of seasonal peaks may be compared directly, regardless of the scale used; this is true within and among host species should such a comparison be desired. Furthermore, the work by Rodríguez and Navas (1995) combined with those of Choi et al. (1989), Gauthier and Fisher (1990), Bushek et al. (1994) suggest that the same scale can be confidently applied to other species. However, interpreting the impact of infection intensities on different hosts in different systems requires additional validation. To venture further in making comparisons among studies using different scales, some conversions can be made (Table 4). For example, because Quick (1972) simply reassigned the values used for the descriptive ordinal categories of the Mackin scale, it is a relatively simple matter to convert Mackin scale scores to those of Quick (1972, Table 4), and to then recalculate any summary statistics. When only summary statistics are available, however, the conversion is not as simple. Fig. 9 shows WP data collected from Delaware Bay oysters between 1990 and 2013 calculated from Mackin scale rankings along the x-axis, and Quick scale rankings along the y-axis. Note that there is an inflection point at 1 where values 61 are doubled, while those >1 are increased by adding 1. A direct conversion of the WP values analogous to a direct conversion of individual scores is not valid, because multiple distributions of data can yield a given WP (see Fig. 6). Some WPs may include data on both sides of the inflection point while others do not. This effect is greatest around the inflection point (Fig. 9). A second-order polynomial forced through the origin fits the data well, and may be used to convert WPs from one scale to the other. Raw data using the other scales are necessary to generate conversions similar to that shown in Fig. 9. Equations to convert summary statistics among other scales can be crudely estimated by plotting the relationships in Table 4,

Fig. 9. Weighted prevalence (WP) data from Delaware Bay, NJ. Each point represents a separate sample for which WP was calculated separately using the Mackin scale (x-axis) and the Quick scale (y-axis). The regression line represents a second order polynomial forced through the origin because zero represents uninfected individuals on both scales: y = 0.0879x2 + 1.589x, R2 = 0.99685.

but the tendency to over- or under-estimate the true value will be unknown until raw data are used to generate relationships, similar to those of Fig. 9. 5. Additional applications for RFTM 5.1. In vitro isolate propagation and manipulation Although P. marinus was first successfully propagated in vitro directly from infected oyster tissues (La Peyre et al., 1993; Gauthier and Vasta, 1993; Kleinschuster and Swink, 1993), RFTM- and ARFTM-enlarged Perkinsus sp. cells are also frequently used as inocula for proliferative Perkinsus sp. in vitro cultures (La Peyre and Faisal, 1995; Bushek and Allen, 1996; Burreson et al., 2005; Casas et al., 2005; Dungan and Reece, 2006; Arzul et al., 2012; da Silva et al., 2013). Several pragmatic advantages encourage the use of RFTM-enlarged Perkinsus sp. cells as in vitro isolate culture inocula (La Peyre and Faisal, 1995; La Peyre, 1996). Some protistan associates of infected mollusc tissues, including ciliates and flagellates, are apparently selectively killed or inhibited by exposure to RFTM or ARFTM media for 48–72 h. Enlarged Perkinsus sp. hypnospores are conveniently identified microscopically during rapid screening of large numbers of separate tissue samples to identify optimum inocula for direct harvest and sub-culture in propagation media. Such assays efficiently identify useful Perkinsus sp. isolate inocula when small tissue samples are incubated in 1.5–2.0 ml of ARFTM in wells of covered, sterile 24-well culture plates; which are conveniently analyzed unstained with an inverted microscope (Dungan and Reece, 2006). 5.2. In vitro manipulations with RFTM to stimulate zoosporulation For wholesale induction of zoosporulation and zoospore release among some Perkinsus sp. in vitro isolates, a brief (48 h, 27 °C)

Fig. 10. Wholesale zoosporulation among Perkinsus chesapeaki in vitro isolate cells following a 48-h exposure to alternative Ray’s fluid thioglycollate medium (ARFTM) before their return to DME: Ham’s F-12 propagation medium. Several enlarged hypnospores with eccentric nuclei bulging into central vacuoles (V) occur among numerous zoosporangia with discharge pores (⁄). Maturing zoosporangia contain 2 or numerous internal zoosporoblast cells that are subdividing toward maturation as infectious, flagellated, motile zoospores with diameters of 2–3 lm. Bar = 30 lm.



transfer of proliferating in vitro trophozoites from DME:Ham’s F-12 Perkinsus sp. propagation medium (DME/F12-3, Burreson et al., 2005) into ARFTM, results in uniform, non-proliferative enlargement of trophozoites. Following return of those ARFTM-enlarged Perkinsus sp. cells to the DME/F12-3 propagation medium for 2–5 d, 70–90% of the enlarged hypnospores undergo classical reductive zoosporulation to yield hundreds of free-swimming flagellated zoospores from each walled zoosporangium. This simple manipulation has induced wholesale zoosporulation among several in vitro isolates of P. chesapeaki, P. olseni, and Perkinsus honshuensis (Dungan and Reece, 2006; Dungan et al., 2007) (Fig. 10). Such manipulations may yield large quantities of motile zoospores for structural, genetic, and functional investigations on several projected roles for zoospores in cell cycles and infectious processes of Perkinsus spp. Induction of widespread zoosporulation is also possible among Perkinsus sp. hypnospores that have been enlarged by RFTM-incubations of infected host tissues. Motile zoospores produced by such in vitro manipulations have been used extensively to transmit experimental infections among and between mollusc hosts (Perkins and Menzel, 1966; Goggin et al., 1989; Waki et al., 2012). In complex applications to insure or restore their natural infectivity and virulence, axenic in vitro Perkinsus sp. isolates have been passaged through uninfected host clams before incubation of tissues from those experimentally infected clams in RFTM to induce zoosporulation as a source of infectious Perkinsus sp. zoospores for disease transmission experiments (Shimokawa et al., 2010; Waki and Yoshinaga, 2013). Likewise, axenic in vitro Perkinsus sp. isolate cells have been passaged through uninfected host clams and then enlarged by RFTM incubations of infected clam tissues, before re-isolation in vitro to test effects of seawater temperature and salinity on their subsequent zoosporulation and proliferation (Umeda et al., 2013). 5.3. PCR assays of RFTM-assayed tissues RFTM assays detect all (7) fully confirmed 2015-members of the genus Perkinsus, but they do not differentiate those species. Among those Perkinsus spp., several are sympatric in some locations, where they may also share common hosts (Dungan and Reece, 2006; Reece et al., 2008; Arzul et al., 2012; da Silva et al., 2014). Where biosecurity or research requirements demand differential identification of pathogen species, post hoc analyses may differentiate species-specific DNAs extracted from mollusc tissues that have been tested by RFTM assays. Relatively recent methods describe direct extraction and PCR-amplification of template DNAs from P. marinus-infected oyster tissues in samples analyzed by standard RFTM assays, including those subjected to oxidative staining with Lugol’s iodine (Audemard et al., 2008). This expanded capability offers the potential to follow low-cost screening by generic RFTM assays for Perkinsus sp. parasites, with species-specific PCR-amplification of DNAs from the same assayed tissues containing pathogen cells, or by amplifications by genus-specific PCR followed by amplicon sequencing (da Silva et al., 2014) or RFLP analyses (Abollo et al., 2006; Takahashi et al., 2009) to identify or differentiate Perkinsus species. Due to the potent capabilities for PCR-amplification of minor contaminant DNAs, results from PCR assays performed on DNAs extracted from tissues previously analyzed by RFTM assays will be seriously compromised if such tissue samples are collected or manipulated in any of the many ways that allow contamination of analyzed tissue samples with extraneous DNAs (Bushek et al., 1994). Since standard RFTM assays detect only viable Perkinsus sp. cells present in tested samples, contaminant DNAs alone will not cause false-positive results in RFTM assays. However,

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Perkinsus sp. DNAs contaminating RFTM-assayed samples may certainly confound the accuracy and integrity of results from subsequent PCR assays. 6. Future applications for RFTM methods RFTM assays retain their original value as relatively inexpensive and technically simple assays for detection of the growing number of Perkinsus spp. that infect a similarly expanding number of molluscs worldwide. They require relatively basic laboratory facilities, whose most sophisticated elements include an autoclave for RFTM sterilization and a standard brightfield microscope for analyzing tested samples. As such, there remains strong potential for expanding their use in surveillance and screening of molluscan shellfish for transport and deployment among water bodies. Similarly, RFTM remains an effective means for stimulating zoosporulation for infectivity studies, morphological studies and other applications. RFTM is an effective transport medium for live Perkinsus sp. cells within infected mollusc tissues, which may be used as inocula for propagation of in vitro Perkinsus sp. isolates by remote microbial culture facilities. Finally, since RFTM assays detect only viable Perkinsus sp. cells that enlarge in the nutrient medium, they can be used to test for the presence of viable pathogen cells in samples that have given positive results in PCR screening assays for Perkinsus sp. DNAs. As technological developments continue in cell culture and molecular detection assays, the RFTM assay that was developed somewhat serendipitously more than 60 years ago, remains a valuable assay with a growing number of modern applications. Acknowledgments Dr. Sammy Mehedy Ray (1919–2013) developed the RFTM assay as a WWII-veteran graduate student at Rice University, where he was mentored by luminaries that included Asa Chandler, John Mackin, Albert Collier, and Sewell Hopkins. Sammy Ray was in turn a consummately generous advisor to innumerable students and colleagues during his long and active faculty tenure at Texas A&M University, including us. This review of the many beneficial consequences and extensions of his seminal research and his lucid writing, is dedicated to the shining legacy of Sammy Ray. Stuart Lehmann (Maryland DNR) and Dorothy Howard (NOAA-NOS) identified and secured much of the cited early literature on development of fluid thioglycollate sterility test media, and Carol McCollough (Maryland DNR) imaged Fig. 4. Figs. 5 and 8 are reprinted with generous permissions from their cited original sources. Invaluable and extensive reviews of archival records of the USA National Institutes of Health (NIH) and the Food and Drug Administration (FDA) by Dr. John Swann, Dr. Victoria Harden, and tenacious archivist Barbara Harkins yielded and restored for public access a copy of one of the extensively cited editions of NIH memoranda on sterility test media by the Division of Biologics, which is currently administered by the FDA. Dr. Susan Ford (Professor Emeritus, Rutgers University) collected much of the data used in Figs. 7 and 9 while a tenured faculty member of the Haskin Shellfish Research Laboratory. Many other colleagues cited within the text, as well as others whose work we may have missed, have provided insightful discussions on the benefits and limitations of RFTM in advancing our understanding of Perkinsus spp. parasites. References Abollo, E., Casas, S.M., Ceschia, G., Villalba, A., 2006. Differential diagnosis of Perkinsus species by polymerase chain reaction–restriction length polymorphism assay. Mol. Cell. Probes 20, 323–329.


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