Hematodinium infection seasonality in the Firth of Clyde (Scotland) Nephrops norvegicus population: a re-evaluation.

Hematodinium infections in Norway lobster Nephrops norvegicus from the Clyde Sea area (CSA) population, Scotland, UK, have previously been undetected in summer. This study aimed to establish if the CSA is actually devoid of infected N. norvegicus in this season. Two PCR assays, an ELISA and 2 tests that detect only patent infection (pleopod and body colour methods) were applied in a 21 mo study. Patent infection was seasonal, appearing predominantly in spring, while subpatent infection diagnosed by ELISA and PCR was highly prevalent in all seasons. Generalised linear modelling supported this assertion, as sampling in September and February significantly increased the probability of finding infected N. norvegicus (p < 0.01); infections were predominantly subpatent and patent respectively, at these times. Therefore, Hematodinium seasonality in N. norvegicus populations is likely to have been an artefact of insensitive diagnostic tests. Light Hematodinium infections were found using PCR assays when patent infections were at their most prevalent and intense, suggesting that infection develops at different rates in different N. norvegicus individuals and that only a portion of the total number of infected N. norvegicus die within a single year. These new data were added to a long-term data series for the CSA (1990 to 2008), which showed that after an initial 5 yr epidemic period, prevalence stabilised at 20 to 25%. Comparisons with 'susceptible-infected-recovered/removed' (SIR) models suggest that this high prevalence is maintained through high birth rates of susceptible host N. norvegicus.

Extracellular proteases and possible disease related virulence mechanisms of two marine bacteria implicated in an opportunistic bacterial infection of Nephrops norvegicus.

The extracellular products (ECP) secreted by two strains of gram-negative bacteria isolated from Nephrops norvegicus exhibiting signs of an opportunistic bacterial infection were investigated with the objective of understanding their role in the spoilage of host muscle tissue and identifying disease related virulence mechanisms. ECP from Vibrio sp. demonstrated no proteolytic activity. ECP from Pseudoalteromonas sp. (isolate N10) degraded several substrates, including azocasein and host muscle tissue. Proteolytic activity increased with temperature. Substrate-impregnated sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the effect of the isolates' ECP on the molecular weight of proteins derived from abdominal muscle tissue revealed that the ECP of Pseudoalteromonas sp. selectively degraded the myosin heavy chain, troponin-T, troponin-I, paramyosin and several unidentified muscle proteins approximately 110 kDa in size. Topomyosin was also reduced in quantity. Degradation of SDS-PAGE gels impregnated with host muscle proteins, by the ECP of Pseudoalteromonas sp. revealed 3 zones of proteolysis, with estimated molecular weights between 100 and 30 kDa, indicating multiple proteases in the ECP. Through the API ZYM system, both isolates demonstrated strong leucine arylamidase activity, with the Vibrio sp. showing strong acid phosphatase activity. These enzymes have been identified as disease related virulence mechanisms in other bacterial pathogens. There is likely a complex pathway to the final condition, involving virulence factors of other species and the stresses involved in capture and transport.

Idiopathic muscle necrosis in the Norway lobster, Nephrops norvegicus (L.): aetiology, pathology and progression to bacteraemia.

The pathology and progression of idiopathic muscle necrosis (IMN) in Nephrops norvegicus and possible aetiologies have been investigated. Trawl capture, aerial exposure and handling initiate IMN, and the condition can be induced through periods of aerial exposure alone, in the absence of trawling. Within 24-48 h after trawl capture IMN progresses to a multi-species bacterial septicaemia, with moribund animals exhibiting clinical signs. The aetiology of this condition has been examined using molecular (16S rRNA gene sequencing) and biochemical (standard taxonomic assays, Biolog) criteria to characterize bacterial isolates from moribund and healthy animals. Histopathology of the IMN phase reveals a loss of sarcomeric structure with necrotic lesions containing pyknotic nuclei, fragments of myofibrils and connective tissue elements. In the bacterial phase there is extensive loss of abdominal muscle structure, and the presence of rod-shaped Gram-negative bacteria in the degrading tissues. The results demonstrate that the IMN condition is connected to stressful conditions imposed on N. norvegicus, but involves no pathogenic agents. This is followed by an opportunistic bacterial infection that causes further tissue spoilage. It is believed that the primary cause of both IMN and bacteraemia is imposed stress, but they are expressed in different time courses.

Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans.

The Norway lobster Nephrops norvegicus (L.) from the coastal waters of Scotland is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. Methods used to detect infection include a morphological index (pleopod diagnosis) and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes based on Hematodinium ribosomal DNA (rDNA). In the PCR assay, a diagnostic band of 380 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected N. norvegicus. The sensitivity of the assay was 1 ng DNA, which is equivalent to 0.6 parasites. The primer pair also detected Hematodinium DNA in preparations of the amphipod Orchomene nanus, indicating that the amphipod may be infected with the same Hematodinium sp. infecting N. norvegicus. DNA probes detected Hematodinium parasites in heart, hepatopancreas and gill tissues from N. norvegicus, and hepatopancreas and gill tissues from Carcinus maenas, confirming Hematodinium infection in the latter.