Lactococcus garvieae outbreaks in Brazilian farms Lactococcosis in Pseudoplatystoma sp. - development of an autogenous vaccine as a control strategy.

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous-based, whole-cell inactivated vaccine (bacterin) and an oil-adjuvanted bacterin. Their efficacy was evaluated at 30 days post-vaccination (d.p.v.) by challenge with L. garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non-specific immune response were compared amongst treatments. High protection levels (P < 0.05) were achieved with the oil-adjuvanted vaccine with a relative percentage survival value of 81.7% at 30 d.p.v. Additionally, the oil-adjuvanted vaccine increased the immunogenicity of the bacterin as indicated by greater agglutination antibody titres from 15 until 60 d.p.v. This is the first report of a positive effect of vaccine administration on the specific immunity of sorubim, and the study showed that a specific antibody plays an important role in sorubim defence against lactococcosis because the innate immune responses were similar in all of the studied animals. These results demonstrated that oil-adjuvanted vaccine can be an effective alternative for the protection of sorubim from L. garvieae disease.

Natural coinfection by Streptococcus agalactiae and Francisella noatunensis subsp. orientalis in farmed Nile tilapia (Oreochromis niloticus L.).

Streptococcus agalactiae and Francisella noatunensis subsp. orientalis (Fno) are important pathogens for farm-raised tilapia worldwide. There are no reports of coinfection caused by S. agalactiae and Fno in fish. This study aimed to determine the aetiology of atypical mortalities in a cage farm of Nile tilapia and to characterize the genetic diversity of the isolates. Fifty-two fish were sampled and subjected to parasitological and bacteriological examination. The S. agalactiae and Fno isolates were genotyped using MLST and REP-PCR, respectively. Whole-genome sequencing was performed to confirm the MLST results. Seven fish were shown coinfected by S. agalactiae and Fno. Chronic hypoxia and a reduction in the water temperature were determined as risk factors for coinfection. Fno isolates were shown clonally related in REP-PCR. The MLST analysis revealed that the S. agalactiae isolates from seven coinfected fish were negative for the glcK gene; however, these were determined to be members of clonal complex CC-552. This is the first description of coinfection by S. agalactiae and Fno in farm-raised Nile tilapia. The coinfection was predisposed by chronic hypoxia and was caused by the main genotypes of S. agalactiae and Fno reported in Brazil. Finally, a new S. agalactiae genotype with glcK gene partially deleted was described.

Sensitivity and specificity of real-time PCR and bacteriological culture for francisellosis in farm-raised Nile tilapia (Oreochromis niloticus L.).

Despite the worldwide occurrence of Francisella noatunensis subsp. orientalis (Fno) infection in farmed tilapia, sensitivity and specificity estimates of commonly used diagnostic tests have not been reported. This study aimed to estimate the sensitivity and specificity of bacteriological culture and qPCR to detect Fno infection. We tested 559 fish, sampled from four farms with different epidemiological scenarios: (i) healthy fish in a hatchery free of Fno; (ii) targeted sampling of diseased fish with suggestive external clinical signs of francisellosis during an outbreak; (iii) convenience sampling of diseased and clinically healthy fish during an outbreak; and (iv) sampling of healthy fish in a cage farm without a history of outbreaks, but with francisellosis reported in other farms in the same reservoir. The qPCR had higher median sensitivity (range, 48.8-99.5%) than culture (range, 1.6-74.4%). Culture had a substantially lower median sensitivity (1.6%) than qPCR (48.8%) to detect Fno in carrier tilapia (farm 4). Median specificity estimates for both tests were >99.2%. The qPCR is the superior test for use in surveillance and monitoring programmes for francisellosis in farmed Nile tilapia, but both tests have high sensitivity and specificity which make them fit for use in the diagnosis of Fno outbreaks.

Whole-Genome Sequence of Francisella noatunensis subsp. orientalis Strain FNO01 Isolated from Diseased Nile Tilapia in Brazil.

This paper describes the complete genome sequence of Francisella noatunensis subsp. orientalis strain FNO01, which was isolated during the first outbreak of francisellosis in cultured Nile tilapia in Brazil. The genome is composed of a circular chromosome with 1,859,830 bp and a G+C content of ~32%.

New hosts and genetic diversity of Flavobacterium columnare isolated from Brazilian native species and Nile tilapia.

Flavobacterium columnare is responsible for disease outbreaks in freshwater fish farms. Several Brazilian native fish have been commercially exploited or studied for aquaculture purposes, including Amazon catfish Leiarius marmoratus × Pseudoplatystoma fasciatum and pacamã Lophiosilurus alexandri. This study aimed to identify the aetiology of disease outbreaks in Amazon catfish and pacamã hatcheries and to address the genetic diversity of F. columnare isolates obtained from diseased fish. Two outbreaks in Amazon catfish and pacamã hatcheries took place in 2010 and 2011. Four F. columnare strains were isolated from these fish and identified by PCR. The disease was successfully reproduced under experimental conditions for both fish species, fulfilling Koch's postulates. The genomovar of these 4 isolates and of an additional 11 isolates from Nile tilapia Oreochromis niloticus was determined by 16S rRNA restriction fragment length polymorphism PCR. The genetic diversity was evaluated by phylogenetic analysis of the 16S rRNA gene and repetitive extragenic palindromic PCR (REP-PCR). Most isolates (n = 13) belonged to genomovar II; the remaining 2 isolates (both from Nile tilapia) were assigned to genomovar I. Phylogenetic analysis and REP-PCR were able to demonstrate intragenomovar diversity. This is the first report of columnaris in Brazilian native Amazon catfish and pacamã. The Brazilian F. columnare isolates showed moderate diversity, and REP-PCR was demonstrated to be a feasible method to evaluate genetic variability in this bacterium.

Characterization of Weissella ceti infections in Brazilian rainbow trout, Oncorhynchus mykiss (Walbaum), farms and development of an oil-adjuvanted vaccine.

Weissella ceti is an emerging bacterial pathogen that affects rainbow trout, Oncorhynchus mykiss (Walbaum), farms. The aims of this study were to genotype W. ceti strains isolated from distinct geographical origins and to determine the efficacy of an oil-adjuvanted vaccine against the disease. Between 2010 and 2012, outbreaks were recorded in five Brazilian farms, and 34 W. ceti isolates were genetically characterized by repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequences PCR and pulsed-field gel electrophoresis. Two different W. ceti vaccines were tested: an aqueous-based whole-cell inactivated vaccine (bacterin) and oil-adjuvanted vaccine. Their efficacy was evaluated in rainbow trout at 30 and 60 days post-vaccination (d.p.v.). W. ceti was found to be a highly homogeneous population in Brazil, with clonally related genotypes. Oil-adjuvanted vaccine exhibited the best (P < 0.05) protection against disease, reaching relative percentage survival (RPS)values of 92% at 30 and 60 d.p.v. Bacterin resulted in RPS values of 67% and 58% at day 30 and 60, respectively. The oil-adjuvanted vaccine provided effective protection against W. ceti infection in rainbow trout.

Complete Genome Sequences of Fish Pathogenic Weissella ceti Strains WS74 and WS105.

We describe here the genome sequencing and annotation of Weissella ceti strains WS74 and WS105, isolated from diseased rainbow trout in Brazil. The two genomes were sequenced with an Ion Torrent personal genome machine (PGM) using a fragment library. The genomes of strains WS74 and WS105 consist of circular chromosomes 1,389,513 bp and 1,390,396 bp long, respectively, both presenting a G+C content of 40.75%.

Outbreaks and genetic diversity of Francisella noatunensis subsp orientalis isolated from farm-raised Nile tilapia (Oreochromis niloticus) in Brazil.

Francisella noatunensis subsp orientalis (FNO) is an emerging pathogen of warm water tilapia in a number of different countries. The disease caused by this bacterium in fish is characterized by a systemic granulomatous infection that causes high mortality rates during outbreaks. FNO has been previously described in Asia, Europe, and Central and North America. Its occurrence in South America has never been described. Since 2012, outbreaks of a granulomatous disease have been recorded in cage farms of Nile tilapia (Oreochromis niloticus L.) in Brazil. The current study aimed to identify the etiologic agent of recent francisellosis outbreaks at Brazilian tilapia farms, and to characterize the genetic diversity of the pathogen from farms with distinct geographic origins and without epidemiological connections. Bacteriological analysis of 44 diseased Nile tilapia collected from five cage farms in Brazil was performed during 2012 and 2013. The farms were in different locations and had no recent history of animal or biological material transport between each other. Sixty-two FNO isolates were identified on the basis of FNO-specific qPCR. The main predisposing factors for the occurrence of outbreaks on Brazilian farms were lower water temperature (<22°C) and life stage of fish, affecting mainly fry, fingerlings and young adults (live weight <100 g). The genetic diversity of the Brazilian FNO isolates was evaluated using repetitive extragenic palindromic-PCR. The isolates from different origins were shown to be clonally related. This is the first report of the occurrence and genetic diversity of FNO in South America.

Genotyping of Streptococcus dysgalactiae strains isolated from Nile tilapia, Oreochromis niloticus (L.).

Streptococcus dysgalactiae is an emerging fish pathogen that is responsible for outbreaks of disease on fish farms around the world. Recently, this bacterium was associated with an outbreak at a Nile tilapia, Oreochromis niloticus (L.), farm in Brazil. The aim of this study was to evaluate the genetic diversity, best genotyping method and aspects of molecular epidemiology of S. dysgalactiae infections in Nile tilapia farms in Brazil. Twenty-one isolates from four farms located in different Brazilian states were characterized genetically using pulsed-field gel electrophoresis (PFGE), ERIC-PCR, REP-PCR and sodA gene sequencing. The discriminatory power of the different typing methods was compared using Simpson's index of diversity. Identical sodA gene sequences were obtained from all isolates, and ERIC-PCR and REP-PCR were unable to discriminate among the isolates. PFGE typing detected three different genetic patterns between the 21 strains evaluated; thus, it was the best genotyping method for use with this pathogen. The strains from Ceará State were genetically divergent from those from Alagoas State. The S. dysgalactiae isolates analysed in this study constituted a genetically diverse population with a clear association between geographical origin and genotype.

In silico prediction of conserved vaccine targets in Streptococcus agalactiae strains isolated from fish, cattle, and human samples.

Streptococcus agalactiae (Lancefield group B; group B streptococci) is a major pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis and meningitis in humans. The available prophylactic measures for conserving human and animal health are not totally effective and have limitations. Effective vaccines against the different serotypes or genotypes of pathogenic strains from the various hosts would be useful. We used an in silico strategy to identify conserved vaccine candidates in 15 genomes of group B streptococci strains isolated from human, bovine, and fish samples. The degree of conservation, subcellular localization, and immunogenic potential of S. agalactiae proteins were investigated. We identified 36 antigenic proteins that were conserved in all 15 genomes. Among these proteins, 5 and 23 were shared only by human or fish strains, respectively. These potential vaccine targets may help develop effective vaccines that will help prevent S. agalactiae infection.

Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.

Genetic diversity and new genotyping scheme for fish pathogenic Streptococcus agalactiae.

UNLABELLED: This study aimed to assess the genetic diversity of fish isolates of Streptococcus agalactiae by capsular serotyping, MLST and the pattern of selected virulence genes. Forty-six isolates from Nile tilapia and Amazon catfish were screened by PCR for the twelve virulence genes. The molecular capsular type and sequence type (ST) were determined. Two capsular types (Ia and Ib) and four STs (103, 260, 552 and 553) were identified. The ST-552 and ST-553 represent new allelic combinations. Variable results were found for the genes gbs2018-6, lmb, hylB and cylE. The combined evaluation of serotype, sequence type and pattern of the presence or absence of cylE and hylB allowed the classification of isolates into nine genetic profiles (I-IX). The proposed scheme showed higher discriminatory power and was able to detect evolutionary events missed by MLST analysis. This study provides new information about the genetic diversity of fish pathogenic Strep. agalactiae, and the proposed scheme was shown to be an improved approach to genotyping these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that critical genetic events in Streptococcus agalactiae isolates pathogenic for fish have been missed by serotyping and multilocus sequence typing (MLST). A proposed genotyping scheme based on the evaluation of concatenated data from serotyping, MLST, and the presence/absence of virulence genes was created, and this was able to detect old and recent evolutionary events. It provided a better understanding of the genetic diversity of Strep. agalactiae populations from fish and will contribute to future studies of the molecular epidemiology, pathogenesis and evolutionary aspects of this pathogen.

Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD.

The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.

Streptococcus iniae outbreaks in Brazilian Nile tilapia (Oreochromis niloticus L: ) farms

This is the first report of outbreaks of Streptococcus iniae in Nile tilapia farms in South America. Seven isolates were identified by biochemical, serological and molecular tests. Their 16S rRNA gene sequences showed 100% similarity with S. iniae ATCC 29178 and two distinct PFGE patterns were observed for Brazilian isolates.

Weissella sp. outbreaks in commercial rainbow trout (Oncorhynchus mykiss) farms in Brazil.

The genus Weissella contains 14 bacterial species that usually occur in nutrient-rich environments and in fermented foods and beverages. Outbreaks of hemorrhagic septicemia were reported in three commercial rainbow trout (Oncorhynchus mykiss) farms in Brazil in 2008 and 2009. Seventy-seven Gram-positive isolates were obtained from 41 diseased fish from these farms. The bacterial strains were identified as Weissella at the genus level using biochemical tests, Weissella genus-specific PCR, and 16S rRNA sequencing. To evaluate potential routes of infection, rainbow trout juveniles were experimentally infected with the pathogen. In addition, the resistance of the pathogen to five antibiotics was tested, and provisional epidemiological cut-off values were calculated using the normalized resistance interpretation (NRI). All isolates presented similar phenotypic profiles and positive reactions for Weissella genus-specific PCR. The 16S rRNA sequences of the Brazilian strains showed 100% similarity with sequences of Chinese isolates that previously were identified as the first case of Weissella sp. infection in fish. The disease was successfully reproduced in the laboratory by intraperitoneal injection, immersion, and cohabitation between diseased and healthy fish. All isolates were resistant to sulfonamide, and based on NRI analysis, one, two, and three isolates were classified as non-wild-type (NWT) for erythromycin, oxytetracycline, and norfloxacin, respectively. This is the first description of multiple cases of Weissella sp. infection in rainbow trout farms outside of China, of infectious routes for the disease, and of provisional epidemiological cut-off values for resistance of these bacteria to four antibiotics.

Streptococcus iniae outbreaks in Brazilian Nile tilapia (Oreochromis niloticus L:) farms.

This is the first report of outbreaks of Streptococcus iniae in Nile tilapia farms in South America. Seven isolates were identified by biochemical, serological and molecular tests. Their 16S rRNA gene sequences showed 100% similarity with S. iniae ATCC 29178 and two distinct PFGE patterns were observed for Brazilian isolates.

Detection of type III secretion system genes in Aeromonas hydrophila and their relationship with virulence in Nile tilapia.

The goals of this study were to develop a PCR technique to detect ascV and aopB genes from the type III secretion system (T3SS), to evaluate the frequency of these genes in Aeromonas hydrophila strains isolated from diseased fish and from aquaculture environments, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in Nile tilapia. The PCR assay developed here successfully detected the target genes, showing three different profiles for the strains ascV+/aopB+, ascV+/aopB-, and ascV-/aopB-. A higher frequency of ascV+/aopB+ was verified in isolates from diseased fish compared to those from aquaculture environments (P<0.05). Among 64 isolates from diseased fish, ascV+/aopB+ (62.5%) was the most frequent profile (P<0.05) and caused more intensive mortality rates. Environmental strains containing the ascV+/aopB+ profile were less virulent than isolates from clinical cases. These results suggest that the presence of a functional T3SS probably increases the virulence of A. hydrophila. The PCR technique was shown to be a specific and efficient tool for detection of T3SS, and this technique can be used for virulence typing of A. hydrophila isolates.

Aspects of the natural history and virulence of S. agalactiae infection in Nile tilapia.

Streptococcus agalactiae is an emerging pathogen in Nile tilapia (Oreochromis niloticus) worldwide. To investigate aspects of the epidemiology, transmission and virulence of S. agalactiae infections, nine outbreaks of meningoencephalitis and septicemia in Nile tilapia farms in Brazil were analyzed. Records from the outbreaks revealed large variation in the weight of fish affected, high mortality, and disease occurrence at water temperatures above 26 degrees C. S. agalactiae was isolated from diseased fish from all farms, and 29 strains were identified by phenotypic tests and 16S rRNA gene sequencing. Five strains from different geographic origins were selected to determine the 50% lethal dose (LD(50)). All strains were highly virulent; for example, strain SA 20-06 had an LD(50) of 90 bacteria. To investigate S. agalactiae transmission, we conducted cohabitation assays with diseased and healthy fish and fish challenges using an immersion bath or gill inoculation. Strain SA 20-06 was used in all assays. The disease was reproduced with characteristic clinical signs and S. agalactiae was reisolated in all trials. The infection route studies were identified as by direct contact or through the water. In conclusion, S. agalactiae, a major pathogen of Nile tilapia in Brazil, exhibited high virulence, regardless of the geographic origin of the isolated strains.