Outbreaks of furunculosis cause significant losses in salmonid aquaculture worldwide. With a recent rise in antimicrobial resistance, regulatory measures to minimize the use of antibiotics in animal husbandry, including aquaculture, have increased scrutiny and availability of veterinary medical products to control this disease in production facilities. In such a regulatory environment, the utility of autogenous vaccines to assist with disease prevention and control as a veterinary-guided prophylactic measure is of high interest to the producers and veterinary services alike. However, evolving concepts of epidemiological units and epidemiological links need to be considered during approval and acceptance procedures for the application of autogenous vaccines in multiple aquaculture facilities. Here, we present the results of solid-state nanopore sequencing (Oxford Nanopore Technologies, ONT) performed on 54 isolates of Aeromonas salmonicida ssp. salmonicida sampled during clinical outbreaks of furunculosis in different aquaculture facilities from Bavaria, Germany, from 2017 to 2020. All of the performed analyses (phylogeny, single nucleotide polymorphism and 3D protein modeling for major immunogenic proteins) support a high probability that all studied isolates belong to the same epidemiological unit. Simultaneously, we describe a cost/effective method of whole genome analysis with the usage of ONT as a viable strategy to study outbreaks of other pathogens in the field of aquatic veterinary medicine for the purpose of developing the best autogenous vaccine candidates applicable to multiple aquaculture establishments.

Organically modified clinoptilolite (member of the zeolite family of minerals; MinazelPlus^®) feed additive, with an average weight of 30 ± 2 g, was used to prevent mycotoxicosis in Nile tilapia (Oreochromis niloticus) through its supplementation for 42 days to the diet contaminated with multiple mycotoxins: aflatoxin B1 (40 µg/kg), fumonisin B1 and B2 (600 µg/kg), zearalenone (50 µg/kg), and deoxynivalenol (150 µg/kg). The fish were divided randomly into four experimental groups (basal diet control—C; fed 2 g/kg MinazelPlus^®—MZ; fed multiple mycotoxins—MT; and fed a combination of MinazelPlus^® and multiple mycotoxins—MZ + MT). Each group consisted of triplicate aquarium setups, with six fish in each replicate. Sampling was performed in weeks 2, 4, and 6. The lymphocyte count was significantly higher in the MZ group compared with the MT group and the MT + MZ group in week 6. An overall decrease in the neutrophil count was observed in the experimental groups. Histopathological analysis was performed in weeks 2 and 6, revealing significant changes in the liver, intestines, kidney, and spleen of fish from the MT group, while the MT + MZ and MZ groups were similar to the control. The addition of 2 g/kg MinazelPlus^® has the ability to prevent and reduce the adverse effects of chronic exposure to low concentrations of multiple mycotoxins in juvenile Nile tilapia.

Mechanisms of action attributed to feed additives are of continuous research interest, increasing our knowledge about the side effects (direct or indirect) of their application. The primary role of organically modified clinoptilolite is to bind multiple polar and non-polar mycotoxins contaminating the feed and remove them during the digestion process and through feces, therefore preventing adsorption and consequences of mycotoxicosis on fish health. However, it is not fully understood if this binding action can influence bacterial communities in the fish digestive tract and possibly other organs, as well as the aquatic environment. Therefore, in this study, Nile tilapias (Oreochromis niloticus) (average weight: 30 ± 2 g; n = 48) were simultaneously exposed to low-level mycotoxins (AFB1 40 µg/kg, FB 600 µg/kg, ZEN 50 µg/kg, and DON 150 µg/kg) added to diet, with and without supplementation of commercially available organically modified clinoptilolite feed additive (MinazelPlus^®, 2 g/kg). After 42 days of continuous exposure, gill tissue, feces, and water were collected, and DNA was extracted from the samples. Results of RT-PCR analysis have revealed significant changes in microbiomes in fish from different groups, most prominently in mycotoxin-exposed fish. No significant changes were detected in water samples between the control and MinazelPlus^® groups, confirming the safety of MinazelPlus^® for aquatic microbial communities. MinazelPlus^® addition to the mycotoxin spiked diet, stabilized fish natural microbiota, and prevented the disbalance of microbial homeostasis observed in fish exposed to dietary mycotoxins.