Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci

Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow‐growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies.     

Hosts and transmission of the crayfish plague pathogen Aphanomyces astaci: a review

The crayfish plague pathogen, Aphanomyces astaci Schikora, has become one of the most well‐studied pathogens of invertebrates. Since its introduction to Europe in the mid‐19th century, it has caused mass crayfish mortalities, resulting in drastic declines of local populations. In contrast, North American crayfish usually serve as latent carriers, although they may also be negatively affected by A. astaci infections under some circumstances. Recent research benefiting from molecular tools has improved our knowledge about various aspects of A. astaci biology. In this review, we summarize these advances, particularly with respect to the host range and transmission. We highlight several aspects that have recently received particular attention, in particular newly confirmed or suspected A. astaci hosts, latent A. astaci infections in populations of European crayfish, and the relationship between A. astaci genotype groups and host taxa.     

Experimental evaluation of the potential for crayfish plague transmission through the digestive system of warm-blooded predators

The crayfish plague pathogen (Aphanomyces astaci) can be transmitted through the digestive system of fish, but its dispersal through mammalian and bird digestive tracts has been considered unlikely, and direct experimental evidence remains scarce. We present a small-scale transmission experiment with European otter and American mink fed with infected crayfish, and experiments testing survival of cultures of five A. astaci strains at temperatures corresponding to those inside mammal and bird bodies. The pathogen was neither isolated from predator excrements nor transmitted to susceptible crayfish exposed to excrements. In agar-based artificial media, it occasionally survived for 15 min at 40.5°C and for 45 min at 37.5°C, but not so when incubated at those temperatures for 45 min and 75 min, respectively. The five tested strains differed in resistance to high temperatures, two (of genotype groups E and D) being more susceptible than other three (of groups A, B and D). Their survival to some extent varied when exposed to the same temperature after several weeks or months, suggesting that some yet-unknown factors may influence A. astaci resistance to temperature stress. Overall, we support the notion that passage through the digestive tract of warm-blooded predators makes A. astaci transmission unlikely.     

Aphanomyces astaci presence in Japan: a threat to the endemic and endangered crayfish species Cambaroides japonicus?

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Comparative effects of various antibiotics, fungicides and disinfectants on Aphanomyces invaderis and other saprolegniaceous fungi

Fifty-four isolates of various fish-pathogenic and saprophytic fungi were characterized in terms of their susceptibility to three antibiotics (penicillin, streptomycin and oxolinic acid), three fungicides (malachite green, hydrogen peroxide and sodium chloride) and three disinfectants (an iodophore, sodium hypochlorite and a solution of peracetic acid and hydrogen peroxide). Aphanomyces invaderis, the fungus associated with the Asian fish disease epizootic ulcerative syndrome (EUS); other Aphanomyces isolates from the similar conditions redspot disease (RSD) and mycotic granulomatosis (MG); and the crayfish plague fungus, Aphanomyces astaci, were more sensitive to most of the chemical agents than the other fungi tested. Two compounds currently being considered for use in aquaculture, hydrogen peroxide and Proxitane 0510, are shown here to have some potential for fungicidal treatments and disinfection, respectively. The implications of this study with respect to the isolation, treatment and control of A. invaderis are discussed.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

Genotyping of Streptococcus dysgalactiae strains isolated from Nile tilapia,Oreochromis niloticus (L.)

Streptococcus dysgalactiae is an emerging fish pathogen that is responsible for outbreaks of disease on fish farms around the world. Recently, this bacterium was associated with an outbreak at a Nile tilapia, Oreochromis niloticus (L.), farm in Brazil. The aim of this study was to evaluate the genetic diversity, best genotyping method and aspects of molecular epidemiology of S. dysgalactiae infections in Nile tilapia farms in Brazil. Twenty‐one isolates from four farms located in different Brazilian states were characterized genetically using pulsed‐field gel electrophoresis (PFGE), ERIC‐PCR, REP‐PCR and sodA gene sequencing. The discriminatory power of the different typing methods was compared using Simpson's index of diversity. Identical sodA gene sequences were obtained from all isolates, and ERIC‐PCR and REP‐PCR were unable to discriminate among the isolates. PFGE typing detected three different genetic patterns between the 21 strains evaluated; thus, it was the best genotyping method for use with this pathogen. The strains from Ceará State were genetically divergent from those from Alagoas State. The S. dysgalactiae isolates analysed in this study constituted a genetically diverse population with a clear association between geographical origin and genotype.     

The harvest of the freshwater crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> Eschscholtz in Turkey: harvest history,impact of crayfish plague,and present distribution of harvested populations

This review focuses on the present distribution of populations of the crayfish Astacus leptodactylus that are harvested in Turkey. It also examines the history of this harvest and the impact that crayfish plague has had on them. Crayfish plague, caused by the fungus-like organism, Aphanomyces astaci Schikora, 1906, is a severe parasite of freshwater crayfish and has caused a lot of damage to A. leptodactylus populations in Turkey since 1984. Turkey was the largest provider of A. leptodactylus to Western Europe from 1970 (or possibly earlier) until 1986. For example, the peak production was reached in the early 1980s, with over 5,000 tonnes being exported in 1984. On the other hand, as a result of the crayfish plague the harvest of A. leptodactylus was reduced severely in most populations in Turkey after 1985. The harvest was only 320 tonnes in 1991. After the occurrence of crayfish plague in Turkey, in order to increase crayfish production uncontrolled A. leptodactylus stockings have been carried out in many waterbodies throughout Turkey. These introductions have caused an increase in the number of A. leptodactylus populations, but exploitation of A. leptodactylus is still under the pressure of the plague, although there has been a steady increase in crayfish production in recent years. The harvest increased to 2,317 tonnes in 2004. Fortunately, among those populations affected by crayfish plague, large amounts of A. leptodactylus can still be harvested from three lakes, ?znik (Bursa), E?irdir (Isparta) and Çivril (Denizli). Thus, it seems that A. leptodactylus has a degree of resistance to crayfish plague. It is therefore interesting to investigate the resistance of A. leptodactylus caught from these populations to crayfish plague.     

The decline of endangered stone crayfish (Austropotamobius torrentium) in southern Germany is related to the spread of invasive alien species and land‐use change

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Natural history and development of the introduced signal crayfish,Pacifastacus leniusculus,in a small,isolated Finnish lake,from 1968 to 1993

Signal crayfish (Pacifastacus leniusculus) originating from Lake Tahoe, California, were introduced into Karisjärvi, a small (11 ha) lake in central Finland (61° 58'N, 25° 32'E), in 1968 and 1969. Since then, the population has been monitored regularly by trap catches. The stocked signals (3–5 years old) were caught until 1973, by which time they had reached ages of 7–9 years. Catches have increased slowly since the early 1980s, the peak occurring in 1991 at 2.0 individuals per trap per night. The population size was estimated as 60 trappable specimens in 1974, 95 in 1981 and 420 in 1988. The mean density of adult population in suitable biotopes was low, 0.07 specimens per m^–2, or 0.3 per shore metre, in 1988. The slow development of the P. leniusculus population has been attributed to environmental factors, mainly the limited area of good crayfish habitat. More than 90 % of all signal crayfish were caught in one-third of the shore area, i.e. in steeply sloping lake beds suitable for burrowing or rich in shelters such as rocks and submerged trees. The signals avoided flat, soft bottoms. Fifty per cent of the female P. leniusculus matured at 90 mm TL (smallest 64 mm), i.e. half of the females entered the breeding population in the autumn of their fourth year. The mean size of newly hatched (stage 2) juveniles was 9.7 mm TL and of one-summer olds 30.3 mm. The largest specimen trapped measured 159 mm. The signal crayfish imported into Finland and stocked were infected with crayfish plague (Aphanomyces astaci), but no mortality has been recorded. Two Branchiobdellidae (Annelida, oligochaeta) epibiont species new to Finland were imported from North America with P. leniusculus. The continuous occurrence of these commensals in the signal crayfish population indicates that they have adapted to Finnish conditions.     

Dietary calcium requirement of red swamp crayfish (Procambarus clarkia)

To investigate dietary calcium requirement of red swamp crayfish (Procambarus clarkia), six semi‐purified diets were formulated to contain different concentrations of calcium (2.7(control group), 6.1, 11.9, 17.6, 23.5 and 29.1 g/kg calcium). Each diet was hand‐fed to triplicate of 15 crayfish with average initial body weight (6.22 ± 0.87) g for 8 weeks. The results showed that weight gain rate (WGR) significantly increased and feed conversion ratio (FCR) significantly decreased from 11.9 to 23.5 g/kg groups (p < .05). Protease activities in intestine and hepatopancreas and parathyroid hormone concentrations in serum significantly decreased with increasing dietary calcium levels (p < .05), while calcium and phosphorus contents in exoskeleton, calcium content in muscle and calcitonin concentrations in serum significantly increased (p < .05). The activities of lipase and amylase in intestine and hepatopancreas, serum alkaline phosphatase and total vitamin D concentrations in serum had significant increase as dietary calcium content increased up to 11.9–17.6 g/kg (p < .05). The inorganic phosphorus content in 29.1 g/kg group was significantly lower than those in other groups (p < .05). Broken‐line model analysis based on WGR and quadratic curve model analysis based on FCR showed that optimal dietary calcium requirement of red swamp crayfish ranged from 12.7 to 17.1 g/kg.     

Colonization history and human translocations explain the population genetic structure of the noble crayfish (Astacus astacus) in Fennoscandia: Implications for the management of a critically endangered species

1. The noble crayfish (Astacus astacus) is an endangered freshwater species in Europe. The main threat is from lethal crayfish plague, caused by the oomycete Aphanomyces astaci that has been spread over Europe by introduced North American crayfish species, acting as chronic carriers of the disease.
2. Most of the remaining noble crayfish populations are found in the Baltic Sea area, and there is an urgent need to implement conservation actions to slow down or halt the extinction rate in this region. However, limited knowledge about the genetic structure of populations in this area has so far precluded the development of conservation strategies that take genetic aspects into consideration.
3. Key objectives of this large-scale genetic study, covering 77 locations mainly from northern Europe, were to describe the contemporary population genetic structure of the noble crayfish in the Fennoscandian peninsula (Sweden, Norway, and Finland), taking postglacial colonization history into account, and to evaluate how human activities such as stocking have affected the genetic structure of the populations.
4. Analyses of 15 microsatellite markers revealed three main genetic clusters corresponding to populations in northern, middle, and southern Fennoscandia, with measures of genetic diversity being markedly higher within populations in the southern cluster. The observed genetic structure probably mirrors two main colonizations of the Baltic Sea basin after the last glaciation period. At the same time, several deviations from this pattern were observed, reflecting past human translocations of noble crayfish.
5. The results are discussed in relation to the conservation and management of this critically endangered species. In particular, we recommend increased efforts to protect the few remaining noble crayfish populations in southern Fennoscandia and the use of genetic information when planning stocking activities, such as reintroductions following local extinctions.

     

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S‐1 and S‐2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP‐PCR and ERIC‐PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10⁴ and 10⁶ CFU per fish with the S‐1 isolate, while 100% mortality rates were recorded in zebrafish at 10² and 10⁴ CFU per fish with the S‐2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.     

Effects of putative dietary probiotics from the gut of milkfish (Chanos chanos) on the growth performance and intestinal enzymatic activities of juvenile Nile tilapia (Oreochromis niloticus)

Enzymes synthesized by the gut microbiome are recognized as vital for assisting host metabolism. This study was conducted to screen and identify bacterial isolates with enzyme-producing abilities from the intestine of milkfish Chanos chanos. Overall, 114 isolates were collected, of which 58% showed clear zones indicating various enzyme activities via plate-based assays. Specifically, 65 isolates were amylolytic, 1 isolate was cellulolytic, and 18 isolates exhibited both activities. The two most promising isolates—CC8 and CC27—were further subjected to spectrophotometric enzyme assays to determine their amylase, cellulase and protease activities. Results showed that amylase, cellulase and protease activities (6.00 ± 0.01, 0.52 ± 0.02 and 18.62 ± 0.16 U mg^?1 protein, respectively) were higher in CC8 than in CC27. Using 16S rRNA gene sequencing, the isolates were putatively identified as Vibrio sp. and Bacillus cereus respectively. At a suspension (mL) to feed (g) ratio of 3:25, dietary supplementation of putative probiotics was carried out to evaluate the growth performance, survival and enzymatic activities of juvenile Nile tilapia Oreochromis niloticus. Results revealed that probiotic-supplemented fish exhibited better growth performance—final average body weight, weight gain, specific growth rate and feed conversion ratio—along with increased intestinal enzymatic activities compared with the control (p < 0.05). Survival rates ranged from 80% to 90% and were statistically similar in all treatments (p > 0.05). This study concludes that the application of Vibrio sp. CC8 and Bacillus cereus CC27, supplemented either in monostrain or multistrain preparation, can promote growth and enzymatic activities in juvenile Nile tilapia.     

Bacillus subtilis CK3 used as an aquatic additive probiotics enhanced the immune response of crayfish Procambarus clarkii against newly identified Aeromonas veronii pathogen

To identify the aetiology of the disease outbreak in cultured Procambarus clarkii, a dominant bacteria strain from the diseased crayfish hepatopancreas was isolated. It was identified as Aeromonas veronii based on biochemical identification and 16S rDNA sequencing. The symptoms of artificially infected crayfish were similar to those of naturally infected P. clarkii. The A. veronii was sensitive to norfloxacin, ciprofloxacin, ceftazidime, cefuroxime and ceftriaxone antibiotics, while resistant to penicillin, ampicillin, oxacillin and piperacillin. Virulence genes such as the enterotoxin genes (act, alt and ast) and haemolytic toxin genes (hlyA and aerA) were detected. After A. veronii infection, the connections between some liver tubules disappeared, the vacuoles appeared in the brush border and the mitochondria were enlarged. The antagonistic action of previous identified B. subtilis CK3 against A. veronii was also detected. The supernatant of B. subtilis CK3 exhibits a significant bactericidal effect on A. veronii. After B. subtilis CK3 immersion, the antioxidant enzymes and immune-related enzyme activities in hepatopancreas were significantly higher than control. The accumulative mortality caused by infection of A. veronii can be significantly reduced by adding B. subtilis CK3 into the aquatic water. These results demonstrated that B. subtilis CK3 could act as a water additive to improve the immune response of P. clarkii against newly identified A. veronii. The present study will provide a new way for the prevention and control of crayfish bacterial diseases and also provide technical support for the healthy cultivation of P. clarkii.     

Direct visualization of the novel pathogen,Spiroplasma eriocheiris,in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization

Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal‐to‐noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species‐specific oligonucleotide probes utilizing the sequences of 16S‐23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.