Margaret Crumlish

Human Streptococcus agalactiae strains in aquatic mammals and fish

BackgroundIn humans, Streptococcus agalactiae or group B streptococcus (GBS) is a frequent coloniser of the rectovaginal tract, a major cause of neonatal infectious disease and an emerging cause of disease in non-pregnant adults. In addition, Streptococcus agalactiae causes invasive disease in fish, compromising food security and posing a zoonotic hazard. We studied the molecular epidemiology of S. agalactiae in fish and other aquatic species to assess potential for pathogen transmission between aquatic species and humans.MethodsIsolates from fish (n = 26), seals (n = 6), a dolphin and a frog were characterized by pulsed-field gel electrophoresis, multilocus sequence typing and standardized 3-set genotyping, i.e. molecular serotyping and profiling of surface protein genes and mobile genetic elements.ResultsFour subpopulations of S. agalactiae were identified among aquatic isolates. Sequence type (ST) 283 serotype III-4 and its novel single locus variant ST491 were detected in fish from Southeast Asia and shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. The human pathogenic strain ST7 serotype Ia was also detected in fish from Asia. ST23 serotype Ia, a subpopulation that is normally associated with human carriage, was found in all grey seals, suggesting that human effluent may contribute to microbial pollution of surface water and exposure of sea mammals to human pathogens. The final subpopulation consisted of non-haemolytic ST260 and ST261 serotype Ib isolates, which belong to a fish-associated clonal complex that has never been reported from humans.ConclusionsThe apparent association of the four subpopulations of S. agalactiae with specific groups of host species suggests that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. Furthermore, it provides a rational framework for exploration of pathogenesis and host-associated genome content of S. agalactiae strains.

Jellyfish as Vectors of Bacterial Disease for Farmed Salmon (Salmo Salar)

Swarms or blooms of jellyfish are increasingly problematic and can result in high mortality rates of farmed fish. Small species of jellyfish, such as Phialella quadrata (13 mm in diameter), are capable of passing through the mesh of sea cages and being sucked into the mouth of fish during respiration. Results of the current study show that the initial damage to gills of farmed Atlantic salmon, likely produced by nematocyst-derived toxins from the jellyfish, was compounded by secondary bacterial infection with Tenacibaculum maritimum. Results also demonstrate that these filamentous bacteria were present on the mouth of the jellyfish and that their DNA sequences were almost identical to those of bacteria present on the salmon gills. This suggests that the bacterial lesions were not the result of an opportunistic infection of damaged tissue, as previously thought. Instead, P. quadrata is probably acting as a vector for this particular bacterial pathogen, and it is the first time that evidence to support such a link has been presented. No prior literature describing the presence of bacteria associated with jellyfish, except studies about their decay, could be found. It is not known if all jellyfish of this and other species carry similar bacteria or the relationship to each other. Their source, the role they play under other circumstances, and indeed whether the jellyfish were themselves diseased are also not known. The high proteolytic capabilities of T. maritimum mean that partially digested gill tissues were readily available to the jellyfish, which rely heavily on intracellular digestion for their nutrition.

Experimental challenge studies in Vietnamese catfish, Pangasianodon hypophthalmus (Sauvage), exposed to Edwardsiella ictaluri and Aeromonas hydrophila

The two main diseases in the pangasius catfish industry are bacillary necrosis of Pangasianodon (BNP) and motile aeromonas septicaemia (MAS), where the aetiological agents have been identified as Edwardsiella ictaluri and Aeromonas hydrophila, respectively. In this study, apparently healthy Pangasianodon hypophthalmus were exposed to E. ictaluri, A. hydrophila or both bacterial species by intraperitoneal injection or immersion. There were 20 fish per treatment group, and the bacterial isolates used for the study were recovered from natural infections of BNP or MAS in farmed Vietnamese P. hypophthalmus. The results of the experimental infections mimicked the natural disease outbreaks reported from these pathogens in P. hypophthalmus. Furthermore, it was clearly demonstrated that E. ictaluri was only recovered from the fish exposed to the bacterium and not recovered from the animals receiving A. hydrophila.

Genomic comparison of virulent and non-virulent Streptococcus agalactiae in fish.

Streptococcus agalactiae infections in fish are predominantly caused by beta-haemolytic strains of clonal complex (CC) 7, notably its namesake sequence type (ST) 7, or by non-haemolytic strains of CC552, including the globally distributed ST260. In contrast, CC23, including its namesake ST23, has been associated with a wide homeothermic and poikilothermic host range, but never with fish. The aim of this study was to determine whether ST23 is virulent in fish and to identify genomic markers of fish adaptation of S. agalactiae. Intraperitoneal challenge of Nile tilapia, Oreochromis niloticus (Linnaeus), showed that ST260 is lethal at doses down to 10(2) cfu per fish, whereas ST23 does not cause disease at 10(7) cfu per fish. Comparison of the genome sequence of ST260 and ST23 with those of strains derived from fish, cattle and humans revealed the presence of genomic elements that are unique to subpopulations of S. agalactiae that have the ability to infect fish (CC7 and CC552). These loci occurred in clusters exhibiting typical signatures of mobile genetic elements. PCR-based screening of a collection of isolates from multiple host species confirmed the association of selected genes with fish-derived strains. Several fish-associated genes encode proteins that potentially provide fitness in the aquatic environment.

Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomus regius Asso (Perciformes: Sciaenidae)

Marine cultured meagre, Argyrosomus regius Asso, in central and western Greece were affected by an outbreak of systemic granulomatous disease subsequently demonstrated to be nocardiosis. The fish were originally imported as juveniles from hatcheries in France and Italy and on-grown in Greece, the latter also providing broodstock for a small number of local Greek hatcheries for the production of second-generation juveniles. The disease in cage reared fish had been present throughout the year, particularly in the 1+ and 2+ year old fish with a low to variable morbidity and 1-4% total mortality. Multiple lesions were visible externally on the skin of affected fish, with severe ulcerations and necrosis. Internally, multifocal yellowish-white nodules, 0.1-0.5 cm in diameter, were visible on the surface of several internal organs. Histopathology revealed systemic granulomatous inflammation. Fite-Faraco staining clearly demonstrated the presence of Nocardia-like organisms which were Gram-positive, long, rod to beaded filamentous bacteria. Nocardia genus-specific 16s RNA primers NG1 and NG2 were used to generate a 600 bp fragment recovered from affected tissue, confirming the diagnosis of Nocardia spp. To our knowledge, this is the first report of nocardiosis in meagre.

Intraspecific diversity of Edwardsiella ictaluri isolates from diseased freshwater catfish, Pangasianodon hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam

A molecular epidemiology study was conducted on 90 Edwardsiella ictaluri isolates recovered from diseased farmed freshwater catfish, Pangasianodon hypophthalmus, cultured in the Mekong Delta, Vietnam. Thirteen isolates of E. ictaluri derived from diseased channel catfish, Ictalurus punctatus, cultured in the USA were included for comparison. All the E.ictaluri isolates tested were found to be biochemically indistinguishable. A repetitive (rep)-PCR using the single (GTG)(5) primer was shown to possess limited discriminatory power, yielding two similar DNA profiles categorized as (GTG)(5) -PCR group 1 or 2 among the Vietnam isolates and (GTG)(5) -PCR group 1 within the USA isolates. Macrorestriction analysis identified 14 and 22 unique pulsotypes by XbaI and SpeI, respectively, among a subset of 59 E. ictaluri isolates. Numerical analysis of the combined macrorestriction profiles revealed three main groups: a distinct cluster formed exclusively of the USA isolates, and a major and minor cluster with outliers contained the Vietnam isolates. Antibiotic susceptibility and plasmid profiling supported the existence of the three groups. The results indicate that macrorestriction analysis may be regarded as a suitable typing method among the E. ictaluri species of limited intraspecific diversity. Furthermore, the findings suggest that E. ictaluri originating from Vietnam may constitute a distinct genetic group.

Draft Genome Sequence of a Nonhemolytic Fish-Pathogenic Streptococcus agalactiae Strain

Streptococcus agalactiae is a significant Gram-positive bacterial pathogen of terrestrial and aquatic animals. A subpopulation of nonhemolytic strains which appear to be pathogenic only for poikilotherms exists. We report here the first draft genome sequence of a nonhemolytic S. agalactiae isolate recovered from a diseased fish.

Differential characterization of emerging skin diseases of rainbow trout - a standardized approach to capturing disease characteristics and development of case definitions

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Recovery of Aeromonas hydrophila associated with bacteraemia in captive snakes

Captive snakes, that is, a Jamaican boa (Epicrates subflavus) a yellow anaconda (Eunectes notaeus) and a corn snake (Pantherophis guttatus guttatus), died with signs of bacteraemia including the presence of petechial haemorrhages in the mouth and gums and haemorrhages in the lung, spleen and intestines. The abdomen and anus were swollen with bloody-tinged mucus in the colon. Aeromonas hydrophila was recovered in dense virtually pure culture growth from the internal organs. Characterization of the isolates was by phenotyping and sequencing of the 16S rRNA gene (sequence homology of 99% with A. hydrophila) with outputs confirming the identity as A. hydrophila. Pathogenicity experiments confirmed virulence to frogs (Rana esculenta) and rainbow trout (Oncorhynchus mykiss).

First identification of Flavobacterium columnare infection in farmed freshwater striped catfish Pangasianodon hypophthalmus

The bacterium Flavobacterium columnare was recovered and identified as the aetiological agent causing freshwater columnaris infection in farmed striped catfish Pangasianodon hypophthalmus (Sauvage) fingerlings that had suffered high mortality rates within commercial hatchery ponds in Vietnam. The gross clinical signs were typical of columnaris-infected fish. Histological examination found numerous Gram-negative, filamentous bacteria present on the skin, muscle and gill tissues of affected fish. The yellow-pigmented bacteria were isolated and identified as F. columnare using primary, biochemical and PCR methods. An experimental immersion-challenge study with 2 strains was also performed. It fulfilled Kochs postulates and showed a median lethal concentration (LC50) of 4.27 × 105 and 1.66 × 106 cfu ml-1 for the F. columnare strains FC-HN and FC-CT, respectively. To the best of our knowledge this is the first report of freshwater columnaris infection in P. hypophthalmus.