Carlos Augusto Gomes Leal

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.

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 dysgalactiae as an agent of septicaemia in Nile tilapia, Oreochromis niloticus (L.).

Streptococcus dysgalactiae is a Gram-positive coccus commonly associated with mastitis in cattle (Aarestrup & Jensen 1996; Waage, Mork, Roros, Aasland, Hunshamar & Odegaard 1999) and pharyngitis in humans (Fox, Turner & Fox 1993; Williams 2003). Outbreaks caused by this pathogen have been reported in cultured fish, including amberjack, Seriola dumerili (Risso), and yellowtail, Seriola quinqueradiata Temminck & Schlegel, both from the marine environment. The disease caused by S. dysgalactiae in fish is characterized by septicaemia and focal necrosis in the caudal peduncle, with moderate to high mortality rates during outbreaks (Nomoto, Munasinghe, Jin, Shimahara, Yasuda, Nakamura, Misawa, Itami & Yoshida 2004; Nomoto, Unose, Shimahara, Nakamura, Hirae, Maebuchi, Harada, Misawa, Itami, Kagawa & Yoshida 2006). Outbreaks of streptococcosis are common in Nile tilapia, Oreochromis niloticus (L.), farms in Brazil. Streptococcus agalactiae infections have been reported in farms located in several states of the country (Mian, Godoy, Leal, Yuhara, Costa & Figueiredo 2009). In contrast to other streptococcal fish pathogens, reports of disease caused by S. dysgalactiae have been restricted to cultured marine fish in Japan (Nomoto et al. 2004, 2006). There are no reports of infections attributed to this bacterium in other countries outside Japan or in any freshwater fish species. This study thus represents the first isolation and description of S. dysgalactiae infection in a freshwater fish species, Nile tilapia. In October 2007, a disease outbreak in a Nile tilapia farm located in Ceará State, Brazil, was investigated. Ten diseased fish showed clinical signs of septicaemia and subcutaneous abscesses in the caudal peduncle region. Swabs of subcutaneous abscesses, brain and kidney of each fish were sampled aseptically, streaked onto 5% sheep blood agar and incubated at 28 C for 72 h. The colonies obtained were tested for Gram staining, catalase and oxidase production, and haemolysis. Ten isolates were kept in 50% glycerol-brain heart infusion (BHI) stock solution at )80 C until use. The strains were further phenotypically and serologically characterized using the API20 Strep and Slidex Latex Agglutination kits (BioMerieux). Total bacterial DNA of the isolates was extracted using a DNeasy kit (Qiagen). S. dysgalactiae-specific PCR was conducted following Hassan, Khan & Lammler (2003). The 16S rDNA of two representative strains (SD54-07 and SD64-07) was sequenced as well as the 16S–23S rDNA intergenic spacer region (ISR). The 16S rRNA gene was amplified by PCR according to the method of Fox, Yan, Dewhirst, Paster, Shames, Murphy, Hayward, Belcher & Mendes (1995). The 16S23S rRNA ISR was amplified following the protocol of Forsman, Tilsala-Timisjärvi & Alatossava (1997). Journal of Fish Diseases 2011, 34, 251–254 doi:10.1111/j.1365-2761.2010.01220.x

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

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.

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.

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.

Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain

ABSTRACT Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity.

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.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

BackgroundThe White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation.ResultsThe duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR.ConclusionThe standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseous lymphadenitis

Corynebacterium pseudotuberculosis strain VD57 (Cp_VD57), a highly virulent, nonmotile, non-sporulating, and a mesophilic bacterium, was isolated from a goat’s granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. Here, we describe a set of features of the strain, together with the details of its complete genome sequence and annotation. The genome comprises of a 2.5 Mbp long, single circular genome with 2,101 protein-coding genes, 12 rRNA, 49 tRNA and 47 pseudogenes and a G + C content of 52.85 %. Genetic variation was detected in Cp_VD57 using C. pseudotuberculosis strain 1002 as reference, wherein small genomic insertions and deletions were identified. The comparative analysis of the genome sequence provides means to better understand the host pathogen interactions of this strain and can also help us to understand the molecular and genetic basis of virulence of this bacterium.