Guilherme Campos Tavares’s research while affiliated with Federal University of Minas Gerais and other places

Streptococcus agalactiae is an important pathogen responsible for cases of high mortality in farmed and wild fish worldwide. In Brazil, this bacterium has been commonly associated with outbreaks in Nile tilapia farms, but other native fish species are also susceptible. Since floating cages are one of the most common culture systems used in the country, the close contact between farmed tilapia and native fish species presents a risk concerning the transmission of this pathogen. In this study, we characterized a mortality outbreak in free-living trahira and in farmed arapaima, as well as the genetic and antimicrobial susceptibility patterns of the isolates obtained. During the outbreaks, moribund fish were sampled and subjected to bacterial examination, after which the isolates were identified via MALDI-ToF analysis. Genotyping was evaluated using repetitive sequence-based PCR (REP-PCR) and multilocus sequence typing (MLST). Antimicrobial susceptibility was evaluated using disc diffusion assays. In addition, whole genome analysis also was performed. S. agalactiae was identified in all diseased fish, all of which belonged to serotype Ib, however, trahira strains were classified as non-typeable lineages in the MLST assay, while arapaima strains were classified as ST260. These isolates were shown to be similar to the main genotype found in Nile tilapia in Brazil, using REP-PCR, MLST and phylogenomic analysis. The pathogenicity of bacterium was confirmed by Kock’s postulates for both fish species. The antimicrobial susceptibility assay showed variable results to the same antibiotics among the isolates, prompting four of the isolates to be classified as multidrug-resistant. This study represents the first report of a natural outbreak of Streptococcus agalactiae infection in wild trahira and farmed arapaima inhabiting the same aquatic environment with Nile tilapia.

Lactococcus spp. has emerged as a pathogen that is affecting global aquaculture, with L. garvieae, L. petauri, and L. formosensis causing piscine lactococcosis. While antimicrobials are commonly used to treat diseases in aquaculture, reports of antimicrobial resistance in fish isolates are increasing. However, little is known about the susceptibility patterns of Lactococcus spp. strains isolated from native fish species in Brazil. This study aimed to assess the antimicrobial susceptibility of these strains and establish a provisional epidemiological cut-off value for L. garvieae using the normalized resistance interpretation approach. A total of 47 isolates were tested: 17 L. garvieae, 24 L. petauri, and 6 L. formosensis. The isolates were classified as wild-type or non-wild-type (NWT) based on inhibition zone diameters. Isolates classified as NWT for three or more antimicrobial classes were considered multidrug-resistant, and the multiple antibiotic resistance (MAR) index was calculated. The results revealed heterogeneity in antimicrobial resistance profiles, with higher resistance to trimethoprim/sulfamethoxazole and norfloxacin. Resistance to other antimicrobials, including florfenicol and oxytetracycline (approved for use in Brazil), varied according to the bacterial species. Lactococcus petauri (87.5%) and L. formosensis (66.7%) showed the highest multidrug resistance, compared to L. garvieae (11.7%), along with higher MAR index values. These findings suggest that multidrug-resistant strains could pose future challenges in the production of native species, underscoring the need for ongoing monitoring of antimicrobial resistance and responsible use of antimicrobials in aquaculture.

Infections with Flavobacterium oreochromis pose risks to Colossoma macropomum production. Since no treatment exists, prophylaxis through vaccination is the only form for disease control. The enzyme-linked immunosorbent assay (ELISA) is widely used in aquaculture to assess animal immunity and mainly to validate vaccination protocols. The method based on the use of immunoglobulin Y (IgY) is an alternative to consider due to its advantages over antibodies produced by small mammals. This study aimed to standardize an indirect ELISA (iELISA) protocol to validate the immunization of C. macropomum against F. oreochromis. A hen and fish were immunized to obtain anti-C. macropomum and anti-F. oreochromis antibodies, which were used to standardize the test. The iELISA was composed of the inactivated bacteria in the coating step, C. macropomum anti-Flavobacterium oreochromis as the primary antibody, and anti-C. macropomum IgY as the secondary antibody. The results demonstrate that the highest antibody titers were among the immunized groups, which were bacterin + adjuvant and bacterin. The iELISA enabled the detection of antibodies produced by C. macropomum, which were mediated by IgY specificity.

Piscine lactococcosis is a disease caused by Lactococcus garvieae, L. petauri and L. formosensis. It is considered to be an emerging disease and poses a risk to global aquaculture production. Studies of the genetic diversity of Lactococcus spp. strains have epidemiological importance since they provide information on the genetic relationships between isolates and indicate geographic and temporal distribution. The aim of this study was to evaluate the genetic diversity of Brazilian Lactococcus spp. isolates obtained from native fish species using PCR-based DNA fingerprinting techniques. For this, 36 isolates were selected and submitted to gyrB sequencing, molecular serotyping, REP-, BOX-, and RAPD-PCR approaches. The discriminatory power and the congruence between the typing methods were calculated. After the correct taxonomic discrimination of the isolate, a total of 14, 5 and 17 isolates were reclassified as L. garvieae, L. formosensis and L. petauri, respectively. L. garvieae and L. petauri are related to serotype I, while L. formosensis strains were classified as serotype II. The typing methods tested are useful for evaluating the genetic diversity of isolates and a comprehensive indication of the diversity was found. Heterogeneous and homogeneous populations were observed for L. garvieae and L. petauri, respectively. RAPD-PCR demonstrated a greater discriminatory power for L. formosensis and L. petauri, while REP-PCR showed better results for L. garvieae strains, thus these methodologies are recommended for genotyping these bacterial species. In conclusion, Brazilian isolates of Lactococcus spp. obtained from native fish species constitute a genetically diverse population and the techniques used in this study allowed a better genetic characterization of the isolates.

Lactococcosis in fish has been associated with Lactococcus garvieae and the recently described L. petauri . However, the relevance of these emerging fish pathogens to Nile tilapia still requires thorough understanding. This study investigated lactococcosis outbreaks in Nile tilapia on Brazilian farms and characterized the isolates through molecular identification of the bacterial species, multilocus sequence typing (MLST) analysis, virulence to Nile tilapia, and antimicrobial susceptibility. Lactococcosis outbreaks were monitored from 2019 to 2022 throughout Brazil. The outbreaks occurred mainly during warmer months, and co-infections were observed in four farms, whereas concurrent bacterial infections were identified in all farms. Since the sequence of the 16S rRNA was not capable of differentiating between L. petauri and L. garvieae , Lactococcus spp. isolates were identified at the species level using the gyrB gene sequence. In total, 30 isolates were classified as L. petauri and two as L. garvieae . All L. petauri isolates were grouped in ST24, except for one isolate which belonged to the newly described ST47. A new ST was also described for the L. garvieae isolates identified, ST46. Furthermore, L. petauri ST24 and ST47 were characterized as singletons, whereas L. garvieae ST46 was grouped with ST16 and ST17 and formed CC17. For the challenge trial, an L. petauri ST24 isolate was chosen considering that this MLST lineage was the most frequently observed. L. petauri was reisolated from challenged Nile tilapia, confirming the pathogenicity of this bacterium to Nile tilapia. The infection in the fish progressed very rapidly, and within 48 h post-challenge clinical signs and the first mortalities were observed. The estimated LD50 was 5.74 × 10 ³ CFU 15 days post-challenge. Provisional epidemiological cutoff values were determined for L. petauri for six antimicrobial agents from different drug classes. All isolates were characterized as wild type (WT) for neomycin and oxytetracycline, whereas 96.67 % of the isolates were characterized as WT for amoxicillin, erythromycin, and florfenicol, and 83.33 % were WT for norfloxacin. Of the 14 outbreaks analyzed, 12 were caused by L. petauri and two by L. garvieae . The gyrB gene sequence was used to differentiate L. petauri from L. garvieae and allowed for the correct identification of these pathogens. Two MSLT lineages of L. petauri were identified and ST24 was observed in different regions of the country, illustrating a rapid expansion of this bacterial lineage. Highlights of the manuscript Lactococcus petauri is pathogenic to Nile tilapia. The MLST lineage most observed was L. petauri ST24, indicating its adaption to infect Nile tilapia. The analysis of the gyrB gene sequence allowed for the correct identification of L. petauri and L. garvieae .

Growth curve of NCDO 2118 under AN (30°C and 37°C) and NAE (30°C and 37°C) conditions.

Total list of peptide and proteins identified in NAE conditions.

Total list of peptide and proteins identified in AN condition.

Supplementary Tables S1–S9.