Crayfish plague is an emerging disease caused by the oomycete Aphanomyces astaci, a pathogen listed among the 100 World's Worst Invasive Alien Species. It was introduced into Europe in the second half of 19th century from North America and caused collapses of European native crayfish populations. Nowadays, A. astaci is widespread in Europe and has spread also to other parts of the world, threatening all susceptible crayfish of non-North American origin. The aims of this MSc thesis were 1) to provide information about crayfish plague outbreaks from recent years, and by using microsatellite and mtDNA markers reveal A. astaci genotypes involved; 2) to test healthy-looking indigenous crayfish for potential occurrence of chronic infections by A. astaci in Czechia. Six new crayfish plague outbreaks were confirmed from 2016 to 2018, involving at least five distinct pathogen strains. My results provide first evidence of the A. astaci genotype group D causing Astacus astacus and Austropotamobius torrentium mass mortalities in Czechia. MtDNA sequencing revealed two haplotypes of the D haplogroup, indicating two independent sources of infection presumably either from ornamental crayfish or spreading from neighbouring countries. The genotype group A was recorded in two A. astacus mortalities and genotype group...

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 4 uppsatser.

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to European indigenous crayfish species, e.g., the noble crayfish (Astacus astacus). The only way to protect susceptible crayfish species from the disease is to prevent the dispersion of the pathogen to their populations. One of the most important sources of the crayfish plague pathogen in Central Europe is the spiny-cheek crayfish (Orconectes limosus), a species of North American origin, which can carry the parasite in its cuticle for years. Some literature sources claimed that the pathogen dispersion from the American vectors is restricted to periods of moulting or to the time before and after the crayfish death. However, experimental evidence for such hypotheses was lacking. The main aim of my thesis was to test these predictions, and the alternative scenario that the crayfish plague pathogen can be transmitted from the infected spiny-cheek crayfish also in other periods. For this purpose, experiments were set up to investigate A. astaci transmission from infected spiny-cheek crayfish to non-infected spiny-cheek or noble crayfish. As expected, the pathogen was transmitted to noble crayfish much more easily than to the uninfected American host. Nevertheless, we succeeded in the pathogen transmission also among spiny-cheek...

The crayfish plague pathogen, the oomycete Aphanomyces astaci, has been decimating populations of European crayfish species for more than 150 years, and is therefore considered one of the 100 worst world's invasive species. A. astaci is highly specialised for a parasitic life, but it can be isolated from moribund crayfish and grown on synthetic media, as it is the case also for several other oomycetes (chapter 7). The life of A. astaci includes three basic forms: mycelium in host's tissues, and the infective units occurring in water, zoospores and cysts. All North American crayfish species tested so far have shown some resistance to A. astaci, i.e., they could carry the infection for long, serving as vectors of the pathogen. Massive sporulation from infected North American crayfish starts when the host is moulting, stressed, or dying (chapter 4). However, I could show in my experiments that some sporulation occurs even from apparently healthy and non-moulting American crayfish hosting A. astaci, so infected North American crayfish must be considered a permanent source of the infection (chapter 4). Five genotype groups of A. astaci have already been distinguished. Strains from a particular genotype group probably share the same original host crayfish species of North American origin. Nevertheless, they can...

- ABSTRACT - The crayfish plague pathogen, Aphanomyces astaci, is responsible for substantial declines and local extinctions of native European crayfish populations. As a consequence, the pathogen is now listed among 100 world's worst invasive alien species. The spread of A. astaci is greatly facilitated by its natural hosts, North American crayfish, that thanks to a long co-evolutionary history with the crayfish plague pathogen evolved efficient defence mechanisms. In contrast, European, Australian and Asian crayfish species are highly susceptible to this disease agent. However, progress of A. astaci infection in native European crayfish was observed to differ between distinct pathogen strains, indicating variability in their virulence. Indeed, we demonstrated a relationship between patterns in crayfish immune response and A. astaci virulence in an experimental infection involving the European noble crayfish and three differently virulent crayfish plague strains. The European continent is currently inhabited by at least eight North American crayfish species. The carrier status was confirmed in six of them, including also Orconectes cf. virilis occurring in the Netherlands and the UK. In this country, we detected Aphanomyces astaci presence in some populations of the non-indigenous crayfish species as well...

<p>The crayfish plague refractory crayfish, <i>Pacifastacus leniusculus</i>, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, <i>Astacus astacus</i>, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established.</p><p>Two serine proteinase genes encoding a subtilisin-like (<i>AaSP1</i>) and a trypsin (<i>AaSP2</i>) enzyme were isolated from the crayfish plague fungus, <i>Aphanomyces astaci</i>. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of <i>AaSP1</i> suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The <i>AaSP2 </i>transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. </p><p>Physiology and genetics of five <i>Aphanomyces</i> strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from <i>A. astaci</i>, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to <i>A. astaci</i> and their name are proposed to be <i>Aphanomyces repetans</i>.</p><p>The crayfish <i>P. leniusculus </i>was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus.</p><p>Two morphotypes of the crayfish parasite <i>Psorospermium haeckeli</i> obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of <i>P. haeckeli</i> are genetically diverse.</p>

The crayfish plague refractory crayfish, Pacifastacus leniusculus, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, Astacus astacus, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established. Two serine proteinase genes encoding a subtilisin-like (AaSP1) and a trypsin (AaSP2) enzyme were isolated from the crayfish plague fungus, Aphanomyces astaci. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of AaSP1 suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The AaSP2 transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. Physiology and genetics of five Aphanomyces strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from A. astaci, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to A. astaci and their name are proposed to be Aphanomyces repetans. The crayfish P. leniusculus was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus. Two morphotypes of the crayfish parasite Psorospermium haeckeli obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of P. haeckeli are genetically diverse.

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Aphanomyces invadans is the pathogen that causes epizootic ulcerative syndrome (EUS), an economically devastating fish disease in southern Asia. The present thesis considered possible improvements to current methods of monitoring EUS, and examined the mechanisms of the host immune response to A. invadans in order to establish whether they could be enhanced to reduce the impact of EUS on aquaculture. Monoclonal antibody (MAb) technology was considered as a possible improvement to the histopathological methods currently used in diagnosis of EUS. Five MAbs were raised to day-old A. invadans germlings. Four gave weak reactions to A. invadans and cross-reacted with other Aphanomyces spp, though they may be useful for future studies on A. invadans. The other, designated MAb 3gJC9, only cross-reacted with the crayfish plague pathogen, A. astaci, and was used for the development of an immunohistochemistry protocol that may be of use in diagnosis. Immunohistochemistry with MAb 3gJC9, which recognised an extracellular product (ECP) of A. invadans, was specific to A. invadans in fish tissue, although it also recognised A. astaci in plague-infected crayfish. It also recognised the mycelium in fish infected with ulcerative mycosis, indicating that ulcerative mycosis is synonymous with EUS. Preliminary observations indicated that both ECPs and what appeared to be a hitherto unreported early stage of the mycelium are important in the pathology of EUS. Studies in vitro on the macrophages of EUS-susceptible giant gourami Osphronemus gouramy and silver barb Barbodes gonionotus, and EUS-resistant Nile tilapia Oreochromis niloticus, found that their macrophages were able to inhibit the growth of A. invadans. The macrophages of striped snakehead Channa striata did not inhibit A. invadans, which may account for their high EUS-susceptibility, especially as A. invadans strongly inhibited the respiratory burst of snakehead macrophages. Studies on humoral immune responses revealed that complement inhibited A. invadans in the case of snakeheads, gourami and barbs but not tilapia or swamp eels Monopterus albus. The humoral responses of the latter were very different to the four other species, and not elucidated. Low levels of anti A. invadans antibodies were found in tilapia and gourami from an EUS-endemic region, and high levels in snakehead. Snakehead antibodies appeared to be able to inhibit A. invadans even when complement was removed, but lower levels were produced at the low temperatures typically associated with EUS. A range of potential immunostimulants were screened for the ability to enhance resistance to EUS. The two successful products were administered as feed supplements to snakeheads and barbs that were subsequently injected intramuscularly with A. invadans. One, the algal extract Ergosan, showed some beneficial effects on snakeheads although the challenge was inconclusive. The other, the vitamin supplement Salar-bec, accelerated the cellular immune response and reduced mortality in snakeheads and barbs, and enhanced antibody production in snakeheads. The antibody response of snakeheads was further studied by comparing the anti- A. invadans antibody level, inhibitory activity of sera in vitro and protective capacity of sera from EUS-naïve snakeheads to that of snakeheads recently exposed to EUS and those subject to long term EUS-exposure. Sera of populations recently exposed to EUS showed an increased level of antibodies, but little improvement in inhibitory or protective activity. Sera from snakeheads that had endured long term exposure showed a wide range of antibody levels, but marked increases in inhibitory and protective activity. Antibodies cross-reacted with non-pathogenic Aphanomyces spp. in all cases.