Genomic comparison of virulent and non-virulent serotype V ST1 Streptococcus agalactiae in fish.

Streptococcus agalactiae or Group B Streptococcus (GBS) is the major pathogen causing pneumonia and meningitis in human, mastitis in dairy cows, and streptococcal disease in tilapia. Previous studies have shown that fish GBS strains are correlated with human GBS strains in evolution and might have cross-host infection ability. Although the invasive disease caused by ST1 GBS in non-pregnant adults and cows is increasing worldwide, infection of fish by ST1 GBS has not been reported. The aim of this study was to determine whether ST1 GBS was virulent in fish and to investigate the genomic characteristics of ST1 GBS strains with different pathogenicity in tilapia. The human-derived serotype V ST1 GBS strains NNA048 and NNA038 were used to intraperitoneally challenge Nile tilapia (Oreochromis niloticus) with doses of 1.0×109CFU/fish, 1.0×107CFU/fish, and 1.0×105CFU/fish, respectively. The cumulative mortality rates of NNA048 infection at three different doses were 100.00%, 83.33%, and 40.00%. In contrast, there were no any sick or dead fish in NNA038 infection group. Histopathological results indicated that challenge of tilapia with NNA048 caused different degree of degeneration and necrosis in brain, liver, spleen, head kidney, and gut, and a large number of blue-stained Streptococcus granules were observed in the tissues. In contrast, there were no any lesions in the tissues of tilapia that were challenged with NNA038. Genome comparison showed that the major genome differences between NNA048 and NNA038 were attributed to the different phage sequences, and there was a 49.8kb length, intact phage sequence encoding 68 proteins in NNA048 genome. SNV and Indels analysis between NNA038 and NNA048 genomes indicated that there were a total of 96 SNVs, 5 deletions and 1 insert. Taken together, serotype V ST1 GBS was comprised of virulent and nonvirulent strains to tilapia, and gene rearrangement might be the main reason of causing different levels of virulence between strains.

Streptococcus agalactiae isolates of serotypes Ia, III and V from human and cow are able to infect tilapia.

Recent studies have shown that group B streptococcus (GBS) may be infectious across hosts. The purpose of this study is to investigate the pathogenicity of clinical GBS isolates with serotypes Ia, III and V from human and cow to tilapia and the evolutionary relationship among these GBS strains of different sources. A total of 27 clinical GBS isolates from human (n=10), cow (n=2) and tilapia (n=15) were analyzed using serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Among them, 15 isolates were tested for their pathogenicity to tilapia. The results showed that five human GBS strains (2 serotype III, 2 serotype Ia and 1 serotype V) infected tilapia with mortality rate ranging from 56.67% to 100%, while the other five human GBS strains tested were unable to infect tilapia. In addition, two cow GBS strains C001 and C003 of serotype III infected tilapia. However, they had significantly lower pathogenicity than the five human strains. Furthermore, human GBS strains H005 and H008, which had very strong ability to infect tilapia, had the same PFGE pattern. MLST analysis showed that the five human and the two cow GBS strains that were able to infect tilapia belonged to clonal complexes CC19, CC23 and CC103. The study for the first time confirmed that human or cow GBS clonal complexes CC19, CC23 and CC103 containing strains with serotypes Ia, III and V could infect tilapia and induce clinical signs under experimental conditions.

Screening vaccine candidate strains against Streptococcus agalactiae of tilapia based on PFGE genotype.

The immunogenicity identification of epidemic strain is important for the development and application of vaccine. In this study, 85 Streptococcus agalactiae prevalent strains from the tilapia main cultured areas of China were distributed among 10 distinct PFGE genotypes (A-J). For each genotype, one representative strain (S.a(A)-S.a(J)) was selected to develop an inactivated whole-cell bacterial vaccine (V(A)-V(J)), which then underwent a protective immunity test. V(A)-V(J) showed similar relative percent survival (RPS) to the homologous or heterologous strains with the identical genotype, while the average RPS among V(A)-V(J) protecting against itself genotype strains showed large differences (44.71-98.81%). The RPS of V(A)-V(J) vaccinated fish against infections by the mixture of S.a(A)-S.a(J) at 15 days post vaccination (dpv) was ranged from 13.33% to 60.00%, and V(B), V(D), V(F), and V(G) showed the highest RPS of 60.00%, 46.67%, 53.33% and 60.00% respectively. V(B), V(D) and V(G) have their own specific protection scope, V(B) showed strong protective immunity to infections caused by A-D, F, G and J (53.57-100%), and V(G) showed strong protective immunity to C-H and J (50.00-100%), whereas V(D) showed weak protective immunity to all non-self genotype strains (14.81-36.67%). The results of the combined vaccination showed that V(G)+V(B) group had wider protection scope and higher RPS value than V(G)+V(D) group. Our results demonstrated that the protective immunity of S. agalactiae from tilapia was not only associated with their serotypes, but also related to their PFGE genotypes. It is difficult to acquire a single vaccine candidate strain that can protect against all genotype strains from the same serotype.

PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China.

Large-scale streptococcal outbreaks occurred continuously in tilapia farms of China from 2009 to 2011. The objective of this study was to characterize the prevalent strains of tilapia streptococci from the main cultured areas of China through species specific PCR and pulse field gel electrophoresis (PFGE). A total of 105 prevalent strains were isolated from Guangdong, Guangxi, Hainan and Fujian provinces between 2006 and 2011, 85 of which were identified as Streptococcus agalactiae while the rest were all identified as Streptococcus iniae. The prevalent stains in 2006 and 2007 were S. iniae (94.7%, 18/19), with S. agalactiae account for only 5.3% (1/19); The prevalent strains in 2009 and 2011 however changed to S. agalactiae (97.7%, 84/86), with only 2.3% (2/86) was S. iniae. Of these 105 strains, a total of 13 PFGE types (A-M) were characterized, among which D, F, G and K genotypes were predominant, accounting for 81.90% (86/105). The cluster analysis of PFGE electropherograms separated S. iniae and S. agalactiae to two distinctive branches, 20 strains of S. iniae exhibiting 3 types of PFGE band patterns with a similarity of 94.8-100%, and the 85 strains of S. agalactiae producing 10 types of PFGE band patterns with a similarity between 48.4% and 100%. Data suggested that the prevalent strains of tilapia streptococci in China have shifted from the former (before 2008) dominant strains of S. iniae to the current (2009-2011) dominant strains of S. agalactiae. Moreover, PFGE genotypes of the prevalent strains demonstrated geographic differences and temporal changes.