were susceptible to the infection, from juveniles of 54 g to
adult fish of more than 1 kg. High mortality rates,
outbreaks at high water temperatures (>27 8C) and
similar clinical features and disease evolution were
observed in all farms. A total of 29 isolates were obtained
and identified as S. agalactiae (Table 2). Considerable
variation in biochemical profile was observed, leading to
classification of the isolates as 13 different phenotypic
variants. More than one pattern was observed for isolates
obtained from farms A–E and I. This was not the case for
farms F, G and H. BLAST analysis of the product sequence
revealed that all strains exhibited similarity of 98% to
previously reported sequences from S. agalactiae. The five
isolates used for LD50 determination caused mortality
rates ranging from 90% to 100%. The first clinical signs
appeared 24 h post-infection, including erratic swimming,
a classic manifestation of brain infection. Mortality
was usually observed 72 h post-infection, but some fish
died only 14 days post-infection. LD50 was only determined
for strain SA 20-06 (6.14 101.17 CFU). However,
the mortality rate was approximately 100% for the other
strains tested at a dilution of 101 CFU. LD50 values could
not be determined for these isolates. In the cohabitation
assay, healthy fish that had direct contact with infected
fish showed clinical signs 24–72 after introduction of the
diseased fish. After 10 days of contact, 100% of the fish
died. In the immersion trial mortality rate of 40% were
observed. The mortality rate reached in the gill inoculation
trial was 33%.