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Yersinia ruckeri sp. nov., the redmouth (RM) bacterium

Cultures of the redmouth (RM) bacterium, one of the etiological agents of redmouth disease in rainbow trout ( Salmo gairdneri ) and certain other fishes, were characterized by means of their biochemical reactions, by deoxyribonucleic acid (DNA) hybridization, and by determination of guanine-plus-cytosine (G+C) ratios in DNA. The DNA relatedness studies confirmed the fact that the RM bacteria are members of the family Enterobacteriaceae and that they comprise a single species that is not closely related to any other species of Enterobacteriaceae. They are about 30% related to species of both Serratia and Yersinia. A comparison of the biochemical reactions of RM bacteria and serratiae indicated that there are many differences between these organisms and that biochemically the RM bacteria are most closely related to yersiniae. The G+C ratios of RM bacteria were approximated to be between 47.5 and 48.5%. These values are similar to those of yersiniae but markedly different from those of serratiae. On the basis of their biochemical reactions and their G+C ratios, the RM bacteria are considered to be a new species of Yersinia , for which the name Yersinia ruckeri is proposed. Strain 2396-61 (= ATCC 29473) is designated the type strain of the species.

International Journal of Systematic Bacteriology↗

Methanolobus taylorii sp nov, a new methylotropic, estuarine methanogen

Previously published phylogenetic studies of 16S rRNA showed that methylotrophic, slightly halophilic, methanogenic strain GS-16 T (T = type strain) represents a new species of bacterium. We propose the name Methanolobus taylorii for this species; strain GS-16 is the type strain.

International Journal of Systematic Bacteriology↗

Desulfuromonas thiophila sp. nov., a new obligately sulfur-reducing bacterium from anoxic freshwater sediment

A mesophilic, acetate-oxidizing, sulfur-reducing bacterium, strain NZ27(T), was isolated from anoxic mud from a freshwater sulfur spring. The cells were ovoid, motile, and gram negative. In addition to acetate, the strain oxidized pyruvate, succinate, and fumarate. Sulfur flower could be replaced by polysulfide as an electron acceptor. Ferric nitrilotriacetic acid was reduced in the presence of pyruvate; however, this reduction did not sustain growth. These phenotypic characteristics suggested that strain NZ27(T) is affiliated with the genus Desulfuromonas. A phylogenetic analysis based on the results of comparative 16S ribosomal DNA sequencing confirmed that strain NZ27(T) belongs to the Desulfuromonas cluster in the recently proposed family 'Geobacteraceae' in the delta subgroup of the Proteobacteria. In addition, the results of DNA-DNA hybridization studies confirmed that strain NZ27(T) represents a novel species. Desulfuromonas thiophila, a name tentatively used in previous publications, is the name proposed for strain NZ27(T) in this paper.

International Journal of Systematic Bacteriology↗

Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the ε-Proteobacteria

Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3(T) and Sulfurospirillum arsenophilum strain MIT-13(T), in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the ?? subclass of the Proteobacteria based on 16S rRNA analysis.

International Journal of Systematic Bacteriology↗

Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes

Kidney and spleen homogenates from each of 60 coho salmon ( Oncorhynchus kisutch ) and steelhead trout ( Salmo gairdneri ) were examined for detection of Renibacterium salmoninarum . The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram-stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis; in steelhead trout, 3% were positive by Gram-stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either coho salmon or steelhead trout by immunodiffusion analysis.

Journal of Wildlife Diseases↗

General and specialized media routinely employed for primary isolation of bacterial pathogens of fishes

There are a number of significant diseases among cultured and free-ranging freshwater fishes that have a bacterial etiology; these represent a variety of gram-negative and gram-positive genera. Confirmatory diagnosis of these diseases involves primary isolation of the causative bacterium on bacteriologic media. Frequently used "general" bacteriologic media simply provide the essential nutrients for growth. For most of the major pathogens, however, there are differential and/or selective media that facilitate primary recovery. Some specialized media are available as "ready-to-use" from suppliers, while others must be prepared. Differential media employ various types of indicator systems, such as pH indicators, that allow diagnosticians to observe assimilation of selected substrates. An advantage to the use of differential media for primary isolation is that they hasten bacterial characterization by yielding the appropriate positive or negative result for a particular substrate, often leading to a presumptive identification. Selective media also incorporate agent(s) that inhibit the growth of contaminants typically encountered with samples from aquatic environments. Media that incorporate differential and/or selective components are ideally based on characters that are unique to the targeted bacterium, and their use can reduce the time associated with diagnosis and facilitate early intervention in affected fish populations. In this review, the concepts of general and differential/selective bacteriologic media and their use and development for fish pathogens are discussed. The media routinely employed for primary isolation of the significant bacterial pathogens of fishes are presented. ?? Wildlife Disease Association 2008.

Journal of Wildlife Diseases↗

An acute septicaemic disease of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar) caused by a Pasteurella-like organism

Ulcerations of the skin associated with haemorrhagic petechiae of liver and kidneys, were the main signs of disease affecting salmon and brown trout in Norway. A death rate of 15–20% was estimated for the 5 month period of mid-March-August, although mortalities occurred throughout the year. Bacteriological examinations, involving 36 isolates, suggested the causual organism to be a Pasteurella although the DNA homology examination gave a G.C. ratio of 55.6 % which is high for Pasteurella as a group.

Journal of Fish Biology↗

Biography of Dr. John L. Fryer

There is probably no one in the fi sh health community who does not recognize the name of Professor John L. Fryer. Active in research for a period of more than 40 years, Dr Fryer has achieved internatio nal acclaim for his wor k on infectious diseases of Pacific salmon . The resea rch from his laboratory at Oregon State University (OSU) has spanned the disciplines of virology, parasitology, bacteriology, cell biology, immuno logy and fi sh physiology, result ing in more than 200 publications, two patents and recognition as one of the world's leading centres for researc h on infec tious diseases of salmon id fi sh.

Journal of Fish Diseases↗

Nature of Aeromonas salmonicida carriage on asymptomatic rainbow trout maintained in a culture system with recirculating water and fluidized sand biofilters

An asymptomatic carrier population of rainbow trout Oncorhynchus mykiss was examined for Aeromonas salmonicida by primary dilution counts on Coomassie Brilliant Blue agar and also by streaking on bacteriological media after a 24–48-h pre-enrichment in tryptic soy broth. The pathogen was detected by primary dilution plate counts in 1 spleen, 15 gill, and 19 mucus samples of the 100 trout examined. Aerornonas samonicida was detected only after 48-h preenrichment in the one spleen that had already tested positive via primary dilution counts. The pathogen was not detected in kidney, liver, and intestinal samples. The occurrence of the pathogen in mucus and gills suggests a predominantly external nature of asymptomatic carriage of A. salmonicida within this population of fish. Repeated examination of fluidized biofilters and tank water showed that A. salmonicida did not become established in the recirculation system.

Journal of Aquatic Animal Health↗

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum , the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Journal of Fish Diseases↗

An alternative bacteriological medium for the isolation of Aeromonas spp.

Two solid bacteriologic media were compared for cultivating Aeromonas spp. from piscine sources: the Rimler-Shotts (RS) medium and a starch-glutamate-ampicillin-penicillin-based medium (SGAP-10C) used for the recovery of Aeromonas spp. from water samples. The selective and differential capacities of the media were assessed March through October 1992 by recovery rate and phenotype of 99 isolates representing 15 genera of bacteria. Recovery frequency of Aeromonas spp. ( n = 62) was similar at 97% on RS and 95% on SGAP-10C. The SGAP-10C medium proved to be more specific than RS toward Aeromonas species (P ≤ 0.005). Use of SGAP-10C at 24 C for 48 hr offers a better choice for the laboratory recovery of Aeromonas spp. from clinical fish specimens.

Journal of Wildlife Diseases↗

Comparison of individual and pooled sampling methods for detecting bacterial pathogens of fish

Examination of finfish populations for viral and bacterial pathogens is an important component of fish disease control programs worldwide. Two methods are commonly used for collecting tissue samples for bacteriological culture, the currently accepted standards for detection of bacterial fish pathogens. The method specified in the Office International des Epizooties Manual of Diagnostic Tests for Aquatic Animals permits combining renal and splenic tissues from as many as 5 fish into pooled samples. The American Fisheries Society (AFS) Blue Book/US Fish and Wildlife Service (USFWS) Inspection Manual specifies the use of a bacteriological loop for collecting samples from the kidney of individual fish. An alternative would be to more fully utilize the pooled samples taken for virology. If implemented, this approach would provide substantial savings in labor and materials. To compare the relative performance of the AFS/USFWS method and this alternative approach, cultures of Yersinia ruckeri were used to establish low-level infections in groups of rainbow trout (Oncorhynchus mykiss) that were sampled by both methods. Yersinia ruckeri was cultured from 22 of 37 groups by at least 1 method. The loop method yielded 18 positive groups, with 1 group positive in the loop samples but negative in the pooled samples. The pooled samples produced 21 positive groups, with 4 groups positive in the pooled samples but negative in the loop samples. There was statistically significant agreement (Spearman coefficient 0.80, P < 0.001) in the relative ability of the 2 sampling methods to permit detection of low-level bacterial infections of rainbow trout.

Journal of Veterinary Diagnostic Investigation↗

Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7. days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes. ?? 2009 University of Cairo.

Journal of Advanced Research↗

Lack of surface-associated microorganisms in a mixed species community of freshwater Unionidae

To determine whether unionids contain surface-attached endosymbiotic bacteria, ciliates, or fungi, we used scanning electron microscopy to examine the epithelial surface of various organs within the digestive systems and mantle cavity of temperate river and lake unionids on a seasonal basis. We also cultured material removed from the lumen of these same organs and from the mantle cavity to detect cellobiose-, cellulose-, and chitin- degrading microbes. No true endosymbiotic fauna were observed attached to the surface of the digestive or mantle tissues of any species of unionid. Microbial growth on cellulose or chitin bacteriological media varied by season and habitat, but not by unionid species or source of the isolate. Lake unionids did not contain microbes capable of digesting cellulose or chitin, whereas unionids from the river site did in March and August, but not in December. Since these cultured cellulose- and chitin-degrading bacteria were never found attached to any unionid tissues, they appeared to be transient forms, not true endosymbionts. Microbes capable of digesting cellobiose were found in every unionid collected in all seasons and habitats, but again, no microbes were directly observed attached to unionid tissues. If unionids, like most other vertebrates, lack digestive enzymes required to initiate primary bond breakage, then the lack of cellulolytic and chitinolytic endosymbionts would affect the ability to utilize cellulose or chitin foods. Thus, in captivity dry feeds based on corn, soybeans, or nauplii should be pre-digested to ensure maximum absorption of nutrients by unionids. The lack of celluloytic or chitinolytic endosymbionts should not affect relocation success, though the seasonal role of transient microbes in unionid nutrition requires further investigation.

Journal of Shellfish Research↗

Biosafety of parenteral Brucella abortus RB51 vaccine in bison calves

Vaccination is considered among the primary management tools for reducing brucellosis prevalence in Greater Yellowstone Area (GYA) ungulates. Before their use, however, vaccine safety and efficacy must be demonstrated. Twenty-seven female bison (Bison bison) calves (approx 5 months old) were vaccinated with Brucella abortus Strain RB51 (1.5 × 1010 colony forming units [CFU], subcutaneously) as part of routine management. We assessed the persistence, pathology, shedding, and transmission associated with RB51 by serial necropsy, bacteriology, histopathology, and serology of 20 of these 27 vaccinated calves, and RB51 serology of 10 nonvaccinated, commingling adult females. With the exception of 1 calf, RB51 dot-blot titers at necropsy were <1:80. Strain RB51 was cultured from lymph nodes in 4 of 4 calves at 14 weeks postvaccination (PV), 4 of 4 calves at 18 weeks PV, 1 of 4 calves at 22 weeks PV, 3 of 4 at 26 weeks PV, and 0 of 4 calves at 30 weeks PV. No gross lesions were observed. Mild histologic changes occurred only in a few draining lymph nodes early in sampling. Adverse clinical effects were not observed in vaccinates. Swabs from nasopharynx, conjunctiva, rectum, and vagina were uniformly culture negative for RB51. Strain RB51 dot-blot assays of bison cows were negative at a 1:20 dilution at 26 weeks PV. Our results suggest that RB51 persists longer in bison calves than in domestic cattle and is systemically distributed within lymphatic tissues. However, bison apparently clear the RB51 vaccine strain without shedding, transmission, or significant adverse reactions.

Journal of Wildlife Management↗

Experimental infection of nontarget species of rodents and birds with Brucella abortus strain RB51 vaccine

The Brucella abortus vaccine strain RB51 (SRB51) is being considered for use in the management of brucellosis in wild bison ( Bison bison ) and elk ( Cervus elaphus ) populations in the Greater Yellowstone Area (USA). Evaluation of the vaccine's safety in non-target species was considered necessary prior to field use. Between June 1998 and December 1999, ground squirrels ( Spermophilus richardsonii , n = 21), deer mice ( Peromyscus maniculatus , n = 14), prairie voles ( Microtus ochrogaster , n = 21), and ravens ( Corvus corax , n = 13) were orally inoculated with SRB51 or physiologic saline. Oral and rectal swabs and blood samples were collected for bacteriologic evaluation. Rodents were necropsied at 8 to 10 wk and 12 to 21 wk post inoculation (PI), and ravens at 7 and 11 wk PI. Spleen, liver and reproductive tissues were collected for bacteriologic and histopathologic evaluation. No differences in clinical signs, appetite, weight loss or gain, or activity were observed between saline- and SRB51-inoculated animals in all four species. Oral and rectal swabs from all species were negative throughout the study. In tissues obtained from SRB51-inoculated animals, the organism was isolated from six of seven (86%) ground squirrels, one of six (17%) deer mice, none of seven voles, and one of five (20%) ravens necropsied at 8, 8, 10, and 7 wk PI, respectively. Tissues from four of seven (57%) SRB51-inoculated ground squirrels were culture positive for the organism 12 wk PI; SRB51 was not recovered from deer mice, voles, or ravens necropsied 12, 21, or 11 wk, respectively, PI. SRB51 was not recovered from saline-inoculated ground squirrels, deer mice, or voles at any time but was recovered from one saline-inoculated raven at necropsy, 7 wk PI, likely attributable to contact with SRB51-inoculated ravens in an adjacent aviary room. Spleen was the primary tissue site of colonization in ground squirrels, followed by the liver and reproductive organs. The results indicate oral exposure to SRB51 does not produce morbibity or mortality in ravens, ground squirrels, deer mice, or prairie voles.

Colorado, Montana, Wyoming↗

Pathology of brucellosis in bison from Yellowstone National Park

Between February 1995 and June 1999, specimens from seven aborted bison ( Bison bison ) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus . Lesions in the B. abortus -infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus -infected herd in July and August, 1997. Results of the bison and cattle studies were similar.

Wyoming↗