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During extended summer trips (16 days), mid-Atlantic sea scallop ( Placopecten magellanicus ) meats hand-shucked at sea and stowed on ice in linen bags become unmarketable because of a spoilage process called "yellowing." Studies were performed to evaluate spoilage of shucked scallop meats by psychrotrophic bacteria under conditions of simulated and actual commercial stowage, and to examine the effects of various prestowage washes and bag materials on this process. A scallop medium and enumeration method based on measuring bacterial populations at the bag/meat interface were developed for this purpose. Results showed that large populations of psychrotrophic bacteria (> 1010 culturable cells per in2) grew on the meats and meat/bag interfaces at 2°C over a 15-day stowage period. Associated with this growth was a rise in surface pH and the appearance and gradual increase of fluorescence on cut muscle surfaces and bags under long-wave UV illumination. Freshly shucked meat surfaces never fluoresced. Of the various deck treatments and bag materials tested, only treatment with 1.25% lactic acid solution retarded pH and bacterial density increases.

A roll-tube repair-detection procedure was developed to enumerate injured and noninjured cells of Bifidobacterium species from water and food samples. This procedure combined the Virginia Polytechnic Institute and State University's anaerobic roll-tube procedure and the repair-detection technique for detecting stressed cells. Mara and Oragui's human bifid sorbitol agar medium was modified for use in the roll-tube procedure by replacing the indicator bromocresol purple with phenyl red (0.027 g/l), adding 0.0006 g of methylene blue per l, increasing the agar content to 25 g/l and adjusting the pH of the medium to 7.1 ± 0.1. The repair-detection roll-tube technique was shown to recover Bifidobacterium cells significantly (P < 0.01) better than pour plates, using the same medium incubated in anaerobe jars, even when a repair-detection system was used. Most repair in the roll tubes occurred in the first hour. B. adolescentis had a poor survival rate after 96 hours in water. Glucose was substituted for sorbitol in the medium used for enumeration of B. longum added to frozen yogurt, because this organism cannot utilize sorbitol. This medium, when used as part of a repair detection system, significantly (P < 0.01) recovered more cells than anaerobic pour plate techniques and was able to separate Bifidobacterium species and Lactobacillus acidophilus by colony morphology. Bifidobacterium cells were 1 mm or larger, round and yellow, while the L. acidophilus colonies were so small (< 1/4 mm) their detection and enumeration was difficult.

High salinity relay of Eastern oysters (Crassostrea virginica) was evaluated as a post-harvest processing (PHP) method for reducing Vibrio vulnificus. This approach relies on the exposure of oysters to natural high salinity waters and preserves a live product compared to previously approved PHPs. Although results of prior studies evaluating high salinity relay as a means to decrease V. vulnificus levels were promising, validation of this method as a PHP following approved guidelines is required. This study was designed to provide data for validation of this method following Food and Drug Administration (FDA) PHP validation guidelines. During each of 3 relay experiments, oysters cultured from 3 different Chesapeake Bay sites of contrasting salinities (10-21 psu) were relayed without acclimation to high salinity waters (31-33 psu) for up to 28 days. Densities of V. vulnificus and densities of total and pathogenic Vibrio parahaemolyticus (as tdh positive strains) were measured using an MPN-quantitative PCR approach. Overall, 9 lots of oysters were relayed with 6 exhibiting initial V. vulnificus10,000/g. As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the30 MPN/g densities requirements for V. vulnificus after 14 to 28 days of relay. Densities of total and pathogenic V. parahaemolyticus in relayed oysters were significantly lower than densities at the sites of origin suggesting an additional benefit associated with high salinity relay. While relay did not have a detrimental effect on oyster condition, oyster mortality levels ranged from 2 to 61% after 28 days of relay. Although the identification of the factors implicated in oyster mortality will require further examination, this study strongly supports the validation of high salinity relay as an effective PHP method to reduce levels of V. vulnificus in oysters to endpoint levels approved for human consumption.

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans , the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum . Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans , the fish and frog MPM show multiple copies of the insertion sequence IS 2404 . Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish > or = 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained < or = 10(3) mycobacteria g(-1).

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correctclassification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary

Slowly growing, non-pigmented mycobacteria were isolated from striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. Growth characteristics, acid-fastness and results of 16S rRNA gene sequencing were consistent with those of the genus Mycobacterium. A unique profile of biochemical reactions was observed among the 21 isolates. A single cluster of eight peaks identified by analysis of mycolic acids (HPLC) resembled those of reference patterns but differed in peak elution times from profiles of reference species of the Mycobacterium tuberculosis complex. One isolate (M1 75T) was placed within the slowly growing mycobacteria by analysis of aligned 168S rRNA gene sequences and was proximate in phylogeny to Mycobacterium ulcerans and Mycobacterium marinum. However, distinct nucleotide differences were detected in the 16S rRNA gene sequence among M175T, M. ulcerans and M. marinum (99.2% similarity). Isolate M175T could be differentiated from other slowly growing, non-pigmented mycobacteria by its inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase and resistance to isoniazid (1 microg ml(-1)), thiacetazone and thiophene-2-carboxylic hydrazide. Based upon these genetic and phenotypic differences, isolate M175T (=ATCC 700981T =NCTC 13215T) is proposed as the type strain of a novel species, Mycobacterium shottsii sp. nov.

Atlantic menhaden Brevoortia tyrannus develop characteristic skin ulcers in response to infection by the oomycete Aphanomyces invadans. To investigate pathogenicity, we conducted a dose response study. Juvenile menhaden were inoculated subcutaneously with 0, 1, 5, 10, 100, and 500 secondary zoospores per fish and monitored for 37 d post-injection (p.i.). Survival rates declined with increasing zoospore dose, with significantly different survivorship curves for the different doses. Moribund and dead fish exhibited characteristic ulcerous lesions at the injection site starting at 13 d p.i. None of the sham-injected control fish (0 zoospore treatment) died. The LD50 (lethal dose killing 50% of exposed menhaden) for inoculated fish was estimated at 9.7 zoospores; however, some fish receiving an estimated single zoospore developed infections that resulted in death. Menhaden were also challenged by aqueous exposure and confirmed that A. invadans was highly pathogenic by this more environmentally realistic route. Fish that were acclimated to culture conditions for 30 d, and presumably free of skin damage, then aqueously exposed to 100 zoospores ml(-1), exhibited 14% lesion prevalence with 11% mortality. Net-handled fish that were similarly infected had a significantly higher lesion prevalence (64%) and mortality (64%). Control fish developed no lesions and did not die. Scanning electron microscopy of fish skin indicated that zoospores adhered to intact epidermis, germinated and penetrated the epithelium with a germ tube. Our results indicate that A. invadans is a primary pathogen of menhaden and is able to cause disease at very low zoospore concentrations.

The infectivity and role of Aphanomyces invadans in the etiology of skin ulcers in Atlantic menhaden Brevoortia tyrannus were investigated with two laboratory challenges. In the first experiment, Atlantic menhaden received subcutaneous injections with secondary zoospores from one of three cultures of Aphanomyces: WIC (an endemic isolate of A. invadans in Atlantic menhaden from the Wicomico River, Maryland), PA7 (an isolate of A. invadans from striped snakehead Channa striata (also known as chevron snakehead), infected with epizootic ulcerative syndrome from Thailand), and ATCC-62427 (an isolate from Atlantic menhaden from North Carolina). Fish were injected with 1.9 × 102 (WIC-low), 1.9 × 103 (WIC-high), 5.2 × 102 (PA7), or 6.0 × 102 (ATCC-62427) zoospores and held in static water at 23.5°C (6‰ salinity) for 21 d. Both low and high doses of WIC caused incipient, granulomatous lesions after 5 d. Fish injected with the high-dose WIC died within 7 d. All fish injected with the low-dose WIC were dead...

We isolated a Mycobacterium sp. resembling Mycobacterium marinum and M. ulcerans from diseased striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. This isolate may represent an undescribed Mycobacterium species, based on phenotypic characteristics and comparative 16S rRNA gene sequence.

A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described.(14)C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D-[1-(14)C]-glucosamine and allowing for its incorporation into the exoskeleton. The cuticle had a total organic carbon content of 0.48 mg C mg(-1) with a specific radioactivity of 6,356 CPM mg(-1). Glucosamine, i.e., chitin content as determined colorimetrically, was 22% (w/w). Microbial degradation and mineralization rates were assessed in batch culture using(14)C-chitin as substrate and York River water as inoculum. Replicate flasks were sampled daily for enumeration of chitinoclastic bacteria and the radiolabel recovered as particulate(14)C-chitin or(14)CO2. The amount of(14)CO2 generated was directly proportional to the loss of particulate(14)C-chitin, with 96% of the added label recovered as the sum of both phases. The maximum rate of mineralization was 207 mg day(-1) g(-1) seeded(14)C-chitin at 20°C. Highest chitinoclastic bacterial counts corresponded to the period of maximum rate of chitinolysis. It is suggested that the rate of chitin mineralization is limited by exoenzymatic depolymerization and not by chitin concentration.

Densities of mesophilic aeromonads were compared in Virginia freshwater lakes with trophic classifications ranging from mesotrophic to hypereutrophic. Aeromonad concentrations were independent of trophic status and did not correlate statistically with water quality parameters used to measure trophic state (total phosphorus, chlorophyll a and Secchi depth). Bacterial indicators (fecal coliforms and Escherichia coli ) used to assess the public health safety of recreational waters correlated with mesophilic aeromonads only when sewage pollution occurred. Overall ( n = 101–107) there were small negative correlations of aeromonad densities with dissolved oxygen ( r = −0.30, P = r = −0.30, P = 0.05). During frequent sampling from April through October 1991, mean mesophilic aeromonad concentrations ranged from 10 3–4 cells 100 ml −1 with the lowest values measured during mid-summer. Over the course of twelve months, 273 aeromonad isolates were identified as belonging to the following biotypes: A. sobria (54%), A. hydrophila (29%), A. caviae (10%), and undetermined (7%). Seasonally, the frequency of isolation of A. sobria , considered the most pathogenic biotype, increased from 17 to 69% when water temperatures exceeded 20°C in a hypereutrophic lake. These temperatures typically occur during periods of peak recreational usage. Phenotypic characteristics associated with virulence were positive in 70% (hemolysis) and 42% (autoagglutination) of glycerol-stored strains ( n = 85), and in 15% (autoagglutination) of strains immediately processed ( n = 73). S-layer proteins associated with invasiveness were not found in 14 strains which autoagglutinated. To evaluate the public health significance of aeromonads in nutrient enriched lakes, our results demonstrate a need to couple investigations of mesophilic aeromonad ecology with virulence and biotype assessments.

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.

Striped bass ( Morone saxatilis ) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden ( Brevoortia tyrannus ) and bay anchovy ( Anchoa mitchilli ). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

The sensitivity and comparative simplicity of 5N stable isotopic tracer techniques has been used to quantify rates of nitrification in aquatic systems. However, the most commonly used method for recovery of inorganic oxidized nitrogen compounds from aqueous samples, which is based on liquid-liquid partitioning, is time consuming and contamination prone. We describe a solid-phase rapid chromatographic method for recovery of 15NO2− and NO3− produced by nitrification in aqueous samples. Compared to liquid-liquid partitioning, the advantages are significantly reduced processing time and reduced potential for contamination. Typical results are presented for the tidal, freshwater reaches of the James River estuary.

The occurrence of male-specific RNA (FRNA) coliphages, proposed as indicators of enteric viruses, was determined in an estuary subject to nonpoint pollution that included fecal inputs from livestock. A host originally developed for detecting FRNA phages in sewage was applied to water and sediment samples. Phages were enumerated using the host Salmonella typhimurium WG49 containing an Escherichia coli plasmid coding for sex pili, and the female parent strain WG45. FRNA phages and fecal coliforms were enumerated in samples collected seasonally from an estuary and associated feeder streams and densities related to selected environmental parameters. Mean phage densities enumerated on WG49 ranged from < 1 to 50 100 ml−1 water and < 13 to 7200 100 g−1 dry sediment. Examination of 300 phages from estuarine and freshwater samples showed that ⩾ 99% were RNase-resistant, ⩾ 94% were lytic to the female parent salmonella strain (WG45), ⩽ 9% were lytic to male E. coli C3000, and none were lytic to female E. coli C. RNase resistant phages lytic to both salmonella strains were noncontractile flexible tailed phages and those lytic to male salmonella or E. coli hosts were filamentous phages. Electron micrographs of the only RNase-sensitive phage recovered that plaqued only male hosts showed cubic phage particles adsorbed to sex pili. Parallel enumerations of environmental samples on WG45 and WG49 yielded equal or greater phage densities on the former host. Purified phages from these samples were lytic to certain salmonella serovars recovered from the environment but did not cross react with fecal coliform or heterotrophic bacteria isolated from the environment. Although the WG49 host was inappropriate to estuarine and freshwater samples examined because of interference by somatic phages, WG45 and WG49 should be examined as hosts for enumerating salmonella phages. Similarly, the public health significance of somatic phages detected by these hosts should be determined. FRNA phages, with a single exception (1187 samples), were not detected in a condemned shellfish growing area subject to nonpoint pollution. This observation questions the application of FRNA phages as indicators of fecal contamination in waters impacted by diffuse fecal inputs.

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.

Eighty striped bass Morone saxatilis were obtained from Delaware Bay using commercial gill nets set adjacent to Woodland Beach (n = 70) and Bowers Beach (n = 10) in December 2003. Fish were examined for gross lesions. Total lengths (TLs) and eviscerated weights were determined to calculate condition factors (K). Portions of spleens were aseptically harvested for bacterial culture, and portions of spleens, kidneys (anterior and posterior), livers, and gonads were obtained for histological examination. The size distribution of the striped bass was relatively homogeneous; the mean TL was about 600 mm for all samples. Mean K exceeded 0.95 in all samples and was not significantly different (P0.05) among samples. Significant differences in mycobacterial infection prevalence (Por = 0.05) were observed among samples; samples obtained at Woodland Beach (WB) on December 10 (53.8%, n = 13) and December 17 (7.1%, n = 42) exhibited the most striking differences in prevalence. Mycobacterial infection intensity ranged from 1 X 10(2) to 1 X 10(7) colony-forming units per gram of spleen. Acanthocephalan infection prevalence and intensity, non-acid-fast bacterial infection prevalence, and fish sex ratio were also significantly different among the samples (Por = 0.05). Similar to the mycobacterial infections, differences in sex ratio, acanthocephalan infection, and non-acid-fast bacterial infection were observed between the WB samples taken on December 10 and 17. However, no significant associations (P0.05) were observed between sex ratio or these infections and mycobacterial infection. The differences in bacterial and parasite infection prevalence and intensity and fish sex ratio in some samples indicate that these fish had a different history and that the epizootiology of mycobacterial infection in striped bass from Delaware Bay may be relatively complex.

The effects of sunlight and the indigenous microbiota onEscherichia coli survival were examined with membrane diffusion chambers deployed in Chesapeake Bay shellfish growing waters. Chambers, fitted with an “upper” UV and visible light-transmitting copolymer film and “lower” semipermeable polycarbonate membrane, were deployed parallel to the water surface to maximize light exposure. Maximum values of a coefficient, kdens, describing changes in culturable cell densities after exposure to sunlight, were 1.7 h−1 and 0.7 h−1 in preliminary tank and in situ experiments, respectively. Mortality and sublethal stress, the latter measured with an electrochemical detection technique, were largest during the first 4 h of exposure. Owing to the light-attenuating properties of Chesapeake Bay water, light-induced cell mortality was significantly reduced at 0.25 m compared with surface exposed cells, and was undetected at 0.5–1.0 m except during seasons of maximal light penetration. Combined exposure to both sunlight and the autochthonous microbiota yielded significantly greater mortality than for either factor alone.

The pathogenic oomyceteAphanomyces invadansis the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detectA. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n= 50) collected in September 2004 were positive forA. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence thatA. invadansis the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusivelyA. invadans.

Oomycete infections caused by Aphanomyces invadans occur in freshwater and estuarine fishes around the world. Along the east coast of the USA, skin ulcers caused by A. invadans are prevalent in Atlantic menhaden, Brevoortia tyrannus. From laboratory observations low salinities appear crucial to transmission of the pathogen. To better understand aspects of transmission, we characterized sporulation and cyst formation of secondary zoospores of two isolates of A. invadans at different salinities and temperatures. Sporulation occurred only at low salinities. At room temperature (ca. 20-22 C), using "pond water" augmented with artificial sea salts, the endemic strain WIC and the Thailand strain PA7 of A. invadans produced free-swimming secondary zoospores at salinities of 0, 1 and 2 psu (practical salinity unit = per thousand), but not at 4 psu or higher. Secondary zoospores of another species, ATCC-62427 (Aphanomyces sp.), were observed at 1, 2, 4 and 8 psu but not at 0 and 12 psu. Secondary zoospores of all three isolates, especially WIC, were abundant and motile 1-2 d postsporulation. Sporulation was temperature dependent and occurred over a relatively narrow range. No sporulation occurred at 4, 30 or 35 C for either WIC or PA7. For both strains zoospore production within 1-3 d after the initiation of sporulation was more prolific at 25 C than at 20 and 15 C. At 15 C production of zoospores was sustained over 11 d for WIC and 5 d for PA7. At room temperature single WIC secondary zoospores remained motile 12-18 h. Salinities exceeding 4 psu or vigorous shaking caused immediate cyst formation of WIC secondary zoospores. Exposure to menhaden tissue, but not tissues of other fishes to secondary zoospores (WIC), caused rapid (2 h) cyst formation. Cysts were capable of excysting when transferred to 1 psu water within 2-3 h of cyst formation. Cysts that had remained encysted in 6.5 psu for 24 h did not excyst when transferred to 1 psu water. Salinity and temperature requirements for sporulation indicate that juvenile menhaden must acquire infections during rain or in low salinity oligohaline waters.

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 °C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 μg ml−1), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15T, has been deposited in the American Type Culture Collection as ATCC BAA-883T and the National Collection of Type Cultures (UK) as NCTC 13318T.

Mycobacteriosis in striped bass Morone saxatilis of Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at 23 degrees C. Mycobacteria were recovered from 76% (n = 149/196) of fish examined. Mycobacterial densities exceeded 10(4) colony forming units x g tissue(-1) in 38% of samples (n = 63/168) that were examined using a quantitative approach. The most frequently recovered mycobacterium, present in 57% (n = 109/192) of characterized samples, was the recently named new species Mycobacterium shottsii. Polyinfections of M. shottsii and other mycobacteria were observed in 25% of samples (n = 47/192) with densities of M. shottsii usually 1 or more orders of magnitude higher than co-isolate(s). Other mycobacteria recovered included isolates that, based on phenotypic traits, resembled M. interjectum, M. marinum, M. scrofulaceum, M. szulgai and M. triplex. M. marinum, commonly associated with fish mycobacteriosis and human disease, was recovered infrequently (3%, n = 6/192). The presence of multiple mycobacterial types occurring at high densities suggests that a variety of mycobacteria could be causative agents of mycobacteriosis in striped bass from the Chesapeake Bay. Striped bass is the major recreational fish species in the Chesapeake Bay, and the significance of the current epizootic to human health and the potential adverse effects on fish stocks are not known.

Striped bass Morone saxatilis were infected intraperitoneally with approximately 10(5) Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21 degrees C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 10(9) colony-forming units g(-1). Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii- and M. gordonae-infected fish resolved over time, and no reactivation of disease was observed.

This chapter focuses on the detection and enumeration topics related to the sanitary microbiology of the receiving waters. The chapter also discusses the examples of alternate indicators. In recent years, the environmental pathogens, such as mycobacteria and Campylobacter , have been the cause for public health concern. Even though clostridia are fermentative saprophytes, they are commonly associated with the food-borne diseases in humans—owing to their ubiquity, the high temperature tolerance of their spores, and their ability to elaborate extremely potent endotoxins. Because Cryptosporidium oocysts are resistant to chlorination, it is suggested that the C. perfringens shouldbe used as an indicator of the drinking water treatment. C. jejuni is most frequently associated with the campylobacteriosis infections in humans. Recovery of cells from the natural waters is hindered by slow growth rates, competitive background microbiota low numbers, and possible non-culturability. Due to the complexities for detection of Campylobacter spp. from environmental samples, many applications of molecular methods based on PCR exist for its detection in poultry, feces, environmental waters, and sewage.

The toxic dinoflagellate, Pfiesteria piscicida, is widely blamed for adverse human health effects, acute fish kills and skin lesion events in fishes, particularly menhaden, Brevoortia tyrannis, inhabiting coastal waters from Delaware to North Carolina, USA. In response, we initiated studies to clarify the etiology and pathogenesis of presumed ‘Pfiesteria-specific’ menhaden skin lesions. Histopathologically, all lesions (>150 fish examined) were associated with a highly invasive and pathogenic fungus eliciting severe tissue necrosis and intense granulomatous inflammation. Severity and extent of the host response indicates that ulcers were at least 1 week old or older. Maryland and Virginia currently use menhaden ulcers as one of several indicators of local Pfiesteria activity. However, their chronic nature, advanced age, and consistent fungal involvement suggest that their use for this purpose may not be valid. We recently isolated an Aphanomyces sp. from the menhaden lesions which by appearance in culture, temperature growth curves, pathogenicity studies in snakehead and positive immunohistochemical staining with polyclonal antibodies suggest the infectious agent is A. invadans (cause of epizootic ulcerative syndrome in Asia, Japan and Australia) or a very closely related species. Ongoing research will address pathogenicity of the fungus in menhaden, genetic comparisons of isolates, and the role of environmental stressors, including P. piscicida, in initiation of the infection.

The coliform group of indicator bacteria has traditionally been the basis for the microbiological growing area standard for shellfish waters since the early 1900s. Since that time researchers have identified deficiencies in its use as an indicator of fecal contamination or potential health risk in aquatic systems (e.g., Berg 1978; Cabelli 1978b; Dutka 1973). Responses to these criticisms are evident in a measured progression to adopt the most fecal-specific coliform organisms as approved indicators. Thus, the fecal coliform has supplanted the total coliform group, and eventually, Escherichia coli may replace the fecal coliform. However, recent studies challenge the fundamental assumption that E. coli is a valid predictor of enteric disease in marine waters (Cabelli et al. 1983). Other workers have identified processes whose effects on indicator densities seriously question the validity of the coliform group as the basis for the growing water standard.

Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors.

Ecotoxicological effects of creosote contamination on benthic bacterial communities in the Elizabeth River, Virginia were investigated using both structural and functional microbial parameters. Parameters included direct counts, viable counts of heterotrophs and “cresol-utilizers”, and bacterial production determined using the tritiated thymidine uptake method. Ancillary data included temperature, salinity, Eh profiles, concentrations of polycyclic aromatic hydrocarbons (PAHs), sediment granulometry and total organic carbon. Two reference stations in relatively nonpolluted areas were sampled for comparative data. Results indicated that cell specific and total heterotrophic bacterial production were depressed in a dose-dependent manner with increasing sediment PAH concentrations. Sediment properties and seasonal changes in temperature appeared to modify the effects of PAHs on bacterial production. Direct bacterial counts and viable counts of total heterotrophs were depressed in the most contaminated sediments. Evidence of creosote adaptation was equivocal, with cresol-utilizer densities not significantly elevated at contaminated stations. The presence of creosote was associated with shifts of Eh toward more negative values compared to nonpolluted sediments. Toxicants which reduce benthic bacterial production may indirectly impact other trophic groups through aberrant cycling of carbon or nutrients. Of the parameters examined, the tritiated thymidine production assay was found to be the most sensitive for detection of ecotoxicological effects.

Experiments designed to measure the oxidation and degradation of crude oils by naturally occurring marine microorganisms are presently being conducted. Fifty active oil degrading cultures have been isolated in enriched seawater containing crude oil. Oil degradation has been determined with gas chromatography, wet combustion, and by measurement of surface tension. Normal paraffin hydrocarbons through C-26 are degraded by two different groups of micro organisms-those growing in the oil phase only and those growing in the aqueous phase. Emulsification of the crude oil through production of surfactants was observed in many of the enriched cultures. Microbial degradation of 35 to 55 per cent of oxidizable crude oil occurred within 60 hours.

Petroleum utilizing microorganisms in flasks containing enriched seawater exhibited a clear metabolic preference for saturated paraffins in a Louisiana crude oil. The rates of oxidation of these compounds were directly proportional to incubation temperature and roughly doubled with a ten degree increase. A pattern of growth consisting of an initially large rate of saturated paraffin oxidation, followed by a decrease and another increase in rate was observed. The initially large rates were attributed to the metabolism of n-paraffins smaller than C-18. No even or odd chain length preference for n-paraffins was indicated. There was no evidence of utilization for aromatic compounds. Application of a microbial culture to an oil slick under simulated field conditions, clearly showed that microbes could accelerate the removal of a Louisiana crude oil from an oil slick on seawater. The rates of oil removal in outdoor, exposed conditions, were twice as large as the rates of evaporative oil loss. The microbes produced a significant change in oil “stickiness”. Measurements indicated the oil was dispersed through microbial activity. The cells preferentially remained at the oil-water interface during the experimental periods.

This paper was prepared for the Second Biennial Symposium on Environmental Conservation presented by the Evangeline Section of the Society of Petroleum Engineers of AIME, to be held in Lafayette, La., Nov. 13–14, 1972. Permission to copy is restricted to an abstract of not more than 300 words. Illustrations may not be copied. The abstract should contain conspicuous acknowledgment of where and by whom the paper is presented. Publication elsewhere after publication in the JOURNAL paper is presented. Publication elsewhere after publication in the JOURNAL OF PETROLEUM TECHNOLOGY or the SOCIETY OF PETROLEUM ENGINEERS JOURNAL is usually granted upon request to the Editor of the appropriate journal provided agreement to give proper credit is made. provided agreement to give proper credit is made. Discussion of this paper is invited. Three copies of any discussion should be sent to the Society of Petroleum Engineers office. Such discussion may be presented at the above meeting and, with the paper, may be considered for publication in one of the two SPE magazines. Abstract Mixed populations of petroleum oxidizing bacteria were isolated from a variety of natural environments. Mixed populations were evaluated for hydrocarbon oxidizing capability (in vitro). In vitro studies employing a nutrient salts enriched seawater indicated utilization of saturated paraffins. n-Paraffins from C-10 to C-30, pristane and phytane were utilized. Experiments with large tanks designed to simulate aspects of the environment revealed that crude oil mass cultured marine bacteria applied to an oil slick in a nutrient salts solution would preferentially remain at the oil-water interface preferentially remain at the oil-water interface while degrading the floating oil slick. A wax-nutrient salts composite is now being considered as a method for retaining a source of nutrient salts at the oil-water interface. Introduction Crude oils are being released into the world ocean in increasing amounts. Besides constituting a needless loss to the world petroleum reserves, the effects of crude petroleum reserves, the effects of crude oils on the marine ecosystem may be undesirable. Although crude oils contain hydrocarbons with demonstrable toxicities and carcinogenic properties in pure form (Blumer, 1969), there are no empirical data describing the responses of biological systems to chronic low-level crude oil pollution. While indirect evidence pollution. While indirect evidence indicates that acutely polluted areas recover from massive petroleum spills (Straughan, 1971; Chan, 1972), hydrocarbons associated with hydrocarbon pollutants have been extracted from exposed marine organisms (Blumer, Souza and Sass, 1970; ZoBell, 1971) Complicating an interpretation of this latter phenomenon is a lack of knowledge describing baseline concentrations and types of hydrocarbons in marine organisms and more significantly, the abilities of organisms to metabolize and/or release hydrocarbons after the pollution scarce has been removed (Lee, Sauerheber, and Benson, 1972). Complementing our ignorance of the biological effects of pollutant crude oils upon marine organisms is the fate of crude oil hydrocarbons introduced into the world ocean.

Three large transite-sided enclosures, constructed in a tidal salt marsh in southeastern Virginia, were utilized to evaluate the effects of crude oil spillage on selected microbial populations. Unweathered Louisiana crude oil was spilled in one enclosure, artificially weathered South Louisiana crude in another, and the third served as a control. Each enclosure was constructed so as to allow unhampered exchange with tidal flow on the tidal creek side. Heterotrophic bacteria and fungi, chitinolytic, cellulytic and petroleum-degrading bacterial populations from the tidal creek, and sediments in intertidal, mid- and back-marsh zones were enumerated at selected intervals following the oil spills. Dominant petroleum-degrading and heterotrophic bacterial isolates were selected for taxonomic grouping. Within several days following the spills, the levels of petroleum-degrading bacteria rose by several orders of magnitude relative to the control enclosure. This differential has been maintained for approximately one year. Plots of the ratio of oil-degrading to heterotrophic bacteria reveal enhancement of the petroleum degrading component of the heterotrophic population. Mean levels of chitinolytic, cellulytic, heterotrophic bacteria and fungi were not statistically different in the oil and control enclosures. The potential significance of these observations is discussed.