Natural outbreaks and molecular characteristics of Streptococcus agalactiae infection in farmed American bullfrog (Rana catesbeiana)

Between June and September 2019, a disease outbreak resulted in high mortality rates on four American bullfrog farms in Wenchang, Hainan Province, China. The main clinical signs included congestion and hemorrhage of the skin on the abdomen and hind limbs, ascites, hepatomegaly and internal organ hyperemia. Gram-positive cocci were isolated from the primary organs and tissues of 149 of 160 collected dead and moribund bullfrogs. The isolates were identified as Streptococcus agalactiae based on their morphology, biochemical characteristics and 16S rDNA sequencing analysis. S. agalactiae was isolated from diseased bullfrogs from all farms, and strain WH914 was selected for experimental infection of bullfrogs. Similar lesions were observed in the experimental bullfrogs, and 17.0%, 33.3% and 100.0% mortality occurred 14 days after administering 8.3 x 10(4) CFU/ml, 8.3 x 10(6) CFU/ml and 8.3 x 10(8 )CFU/ml of the bacteria, respectively. Molecular serotyping of the capsular polysaccharide gene clusters revealed that the S. agalactiae isolates were serotype Ib. The sequence type (ST) of 30 isolates randomly selected from each bullfrog farm was determined by multilocus sequence typing as ST261. Virulence gene profiles for 14 virulence-related genes were constructed via multiplex PCR. All strains contained pavA, sip, iagA, cfb, hylB, bibA, cspA and fbsA but lacked cylE, bac, imb, scpB, rib and bca. S. agalactiae serotype Ib ST261 was isolated from sick or deceased farmed bullfrogs and is recognized as a pathogen of bullfrogs. Natural infections of S. agalactiae serotype Ib ST261 have not been previously reported in American bullfrogs.