Henrique César Pereira Figueiredo

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.

Genotyping of Streptococcus agalactiae strains isolated from fish, human and cattle and their virulence potential in Nile tilapia.

Streptococcus agalactiae (Lancefield group B; GBS) is a pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. The objective of this study was to characterize S. agalactiae isolated from fish (n=27), cows (n=9), and humans (n=10) using pulsed-field gel electrophoresis (PFGE) and to investigate the virulence of the identified strains in Nile tilapia (Oreochromis niloticus). The PFGE types were determined by dendogram analyses and the in vivo virulence was evaluated by experimental infection (using i.p. and immersion routes) of Nile tilapia. Among the fish strains, 5 different PFGE patterns were observed and 21 strains showed the same genetic pattern. In some farms two or three profiles occurred simultaneously. The bovine and human strains exhibited high genetic diversity and few relationships were established among S. agalactiae strains from the three host origins analyzed. Eight S. agalactiae strains from fish caused high mortality of Nile tilapia. Three bovine strains infected Nile tilapia (by i.p. route) and two of those strains caused clinical signs of meningoencephalitis. All human strains (n=5) infected Nile tilapia (by i.p. route) and meningoencephalitis was induced by one strain (by both i.p. and immersion routes). In conclusion, the analyzed strains from the three natural hosts did not show genetic relatedness, yet some of the bovine and human strains were able to infect fish and cause meningoencephalitis. We suggest that genetic linkage is not a prerequisite for S. agalactiae to cross the host-specific barrier.

Freqüência de enteroparasitas em amostras de alface (Lactuca sativa) comercializadas em Lavras, Minas Gerais

The aim of this study was to evaluate the parasitological contamination in samples of lettuce (Lactuca sativa) commercialized in Lavras city, Minas Gerais. The samples of lettuce showed low hygienic conditions, indicated by the presence of parasites of animal or human origin and high concentration of fecal coliforms.

Antibacterial activity of plant extracts from Brazil against fish pathogenic bacteria

The aim of this work was to evaluate the antibacterial activity of Brazilian plants extracts against fish pathogenic bacteria. Forty six methanolic extracts were screened to identify their antibacterial properties against Streptococcus agalactiae, Flavobacterium columnare and Aeromonas hydrophila. Thirty one extracts showed antibacterial activity.

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.

Complete genome sequence of Streptococcus agalactiae strain SA20-06, a fish pathogen associated to meningoencephalitis outbreaks

Streptococcus agalactiae (Lancefield group B; GBS) is the causative agent of meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. Meningoencephalitis is a major health problem for tilapia farming and is responsible for high economic losses worldwide. Despite its importance, the genomic characteristics and the main molecular mechanisms involved in virulence of S. agalactiae isolated from fish are still poorly understood. Here, we present the genomic features of the 1,820,886 bp long complete genome sequence of S. agalactiae SA20-06 isolated from a meningoencephalitis outbreak in Nile tilapia (Oreochromis niloticus) from Brazil, and its annotation, consisting of 1,710 protein-coding genes (excluding pseudogenes), 7 rRNA operons, 79 tRNA genes and 62 pseudogenes.

Perfil de susceptibilidade a antimicrobianos de bactérias isoladas em diferentes sistemas de cultivo de tilápia-do-nilo (Oreochromis niloticus)

Bacterial populations from different tilapia culture system with no antimicrobial use were characterized. Concret pond and commercial feed, land pond and commercial feed, and land pond and animal manure were used in systems I, II and III, respectively. Ninety-eight bacterial strains were subjected to sensitivity testing. Members of Vibrionaceae were the most prevalent in all systems analysed. The most bacterial strains were resistant to ampicillin and erithromicin, but resistance to norfloxacin and gentamicin were uncommon. A half to bacterial isolates was resistant to tetracycline. From the 98 bacterial isolates, 96% were resistant to two or more antimicrobials. The multiple antimicrobial resistance index was determined and it was similar for all systems analyzed (Mar= 0.4), indicating a high risk source for multiple antibiotic resistance.