Microbiological analysis of skin lesions of cod (Gadus morhua) from the southern part of the Baltic Sea
, , , , and | Mar 23, 2024
Published Online: Mar 23, 2024
Page range: 79 - 92
Received: Jul 21, 2023
Accepted: Jan 17, 2024
DOI: https://doi.org/10.2478/jvetres-2024-0004
Keywords
© 2024 Agnieszka Pękala-Safińska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
The Baltic Sea fish population includes more than 100 marine and brackish water species. The low salinity and other specific environmental conditions are conducive to cod (
The accumulation of contaminants in aquatic animals can cause severe stress, resulting in various pathophysiological disturbances in fish. Piscine health is determined by the sum of factors including the state of the immune system, the presence of different pathogens, and the conditions in the aquatic environment. Disruption of the established balance can have serious consequences. One may be skin lesions and ulcers, which often penetrate the muscles and erode fins. In the last decade, Polish fishermen have observed severe health problems in Baltic cod (24). Similar symptoms were also noticed in fish from regions of the Baltic Sea outside the Polish exclusive economic zone (42, 58). In response, recommendations regarding monitoring Baltic cod diseases and parasites and identifying and grading the causative pathogens were formulated (30). They acknowledge the economic losses in the lower cod catch and intend skin lesion inspection to be implemented at all levels, from the fishery personnel to scientists and even the government.
Fish can be affected by various diseases and health disorders exhibiting different stages of development and intensity. Skin ulcers are the most commonly observed among Baltic Sea cod. Bacteria are known as aetiological factors, both primary and secondary, of these clinical signs, complicating previous mechanical irritations of the skin. The observed symptoms include haemorrhages on the fins and the skin’s surface, skin lesions, and ulceration or erosion of the fins. Clinical signs of fish health disorders vary depending on the aetiological agent (24, 30, 42, 58). Previous reports indicated
Therefore, the aim of our study was the analysis of the dynamics of skin lesions observed in Baltic cod in the context of the isolated bacteria which could be their aetiological factors. The results of the analysis were the species of microflora determined to be causing skin lesions in fish from the Polish exclusive economic zone (EEZ) of the southern Baltic Sea caught during two research surveys in 2016 and 2017.
The Baltic cod were collected during two surveys by the Baltica research vessel which took place in April 2016 and January 2017. The explored region was the Polish EEZ of the southern Baltic Sea in the International Council for the Exploration of the Sea Subdivisions 25 and 26. Fish caught in the following five fishing grounds were examined: the Gdansk Bay, Słupsk Furrow, zone near Kołobrzeg-Darłowo and Bornholm Basin north and south parts (Fig. 1). After each haul, cod were measured for body length and their health was examined. The presence of parasites and externally visible disease symptoms, particularly skin lesions and ulceration, was ascertained. The visual disorders observed on the skin were classified according to Bucker
Fig. 1.
Areas where Baltic cod were sampled in 2016 and 2017 within the Polish exclusive economic zone of the southern Baltic Sea (by Podolska M. and Trella K.)
Each fish that exhibited external disorders manifested on its skin was collected for bacteriological examination. Scrapings from the skin lesions/ulceration were taken separately and inoculated onto appropriate media: nutrient agar supplemented with 5% horse blood (BA) (BioMaxima, Lublin, Poland), trypticase soy agar (TSA) (bioMérieux, Marcy-l’Étoile, France),
After Gram staining and morphological assessment of pure bacterial isolates, their biochemical identification was performed using appropriate analytical profile index (API) tests (API 20 E, API 20 NE, API Staph and API Strep) and the VITEK 2 system (bioMérieux), according to the manufacturer’s instructions. The temperature of API test incubation was modified to 22°C ± 2°C.
The 16S rRNA gene was sequenced as described previously (45, 48). The total genomic DNA was isolated from pure bacterial cultures with a DNeasy Blood & Tissue Kit following the producer’s protocol (Qiagen, Hilden, Germany). The quality and concentration of DNA were analysed using a Multiskan GO Microplate Spectrophotometer (Thermo Scientific, Vantaa, Finland). The extracted DNA was used for 16S rRNA gene sequence analysis. Amplification of the V1–V9 regions of the 16S rRNA gene was performed using Eubac27F (5′-AGAGTTTGATC(C/A)TGGCTCAG-3′) and Eubac1492 (5′-TACGG(C/T) TACCTTGTTACGACTT-3′) universal primers as described previously (45, 46, 48). The purified PCR products were sequenced using a 3730xl DNA Analyzer (Life Technologies, Singapore) by Genomed S.A. (Warsaw, Poland) and their sequence data were processed with Molecular Evolutionary Genetics Analysis software (MEGA version 7.0). Phylogenetic analysis of the 16S rRNA gene was performed by the maximum-likelihood method with the Tamura–Nei model using 1,000 bootstrap replicates (55). A tree was created based on the nucleotide sequences of the 16S rRNA gene, which were compared with sequences available in GenBank. For the
Broth microdilution methods provided in the Clinical and Laboratory Standards Institute VET04-A2 guide were used to determine the MICs of antimicrobials against the collected bacterial isolates (16). The selection of bacteria isolates for the MIC analysis was based on the biochemical properties of the microorganisms and represented each of the obtained biochemical profiles. Mueller–Hinton broth (Oxoid, Basingstoke, UK) was used to prepare inoculum, and the incubation condition was set at 22°C for 24 h. No MIC plate designated for bacteria isolated from aquatic animals being on the market, a user-defined POLARGEN Sensititre plate (Trek Diagnostic Systems, East Grinstead, UK) was used. The concentrations of the following antimicrobials representing different prescribing advice categories were selected for analysis: two fluoroquinolones (enrofloxacin at 0.03–16 μg/mL and ciprofloxacin at 0.03–4 μg/mL) and a quinolone (flumequine at 0.25–64 μg/mL) from category B (“Restrict”); an aminopenicillin in combination with a beta-lactamase (amoxicillin and clavulanic acid at 0.12/0.06–16/8 μg/mL) and a phenicol (florfenicol at 0.015–2 μg/mL) from category C (“Caution”); and a tetracycline (doxycycline at 0.5–64 μg/mL), oxytetracycline at 1–128 μg/mL and a sulfonamide (sulphamethoxazole at 8–1024 μg/mL) from category D (“Prudence”) (20). The results of MIC tests described as epidemiological cut-off values establish an isolate as a wild type (WT) or a non-wild type (NWT) (53). A wild-type isolate is defined as one without phenotypically detectable resistance mechanisms, in contrast to an NWT.
Based on the examination of single fish for the presence of pathological changes, the prevalence of each disease in a sample was calculated according to the formula p = x/n, where p is the prevalence, x is the number of fish affected and n is the number of fish examined. The prevalence was expressed as a percentage (p ≤ 100%).
During two research surveys, 1,381 Baltic cod were caught and examined (667 in 2016 and 714 in 2017) in the following fishing grounds: the Gdańsk Bay, Słupsk Furrow, zone near Kołobrzeg-Darłowo, and Bornholm Basin north and south parts. Overall, 164 fish (p = 11.2%) exhibited pathological changes manifested by skin ulceration. The prevalence of diseased fish was p = 15% (98 individuals) in 2016 and 9% (66 individuals) in 2017. Of all the cod fishing grounds, the Gdańsk Bay was the source of the fish with the highest prevalence of skin disorders (p = 64% and p = 45%, respectively in 2016 and 2017) (Figs 2 and 3). The acute stage, characterised by red and open or almost open inflammatory skin lesions (Fig. 4), was observed among 8.5% of the diseased Baltic cod collected from this fishing ground (Fig. 3). The highest prevalence (11.2%) of the skin changes characteristic of the healing process (Fig. 3) was also observed among the cod from the Gdańsk Bay. This group included both scar formation and melanin deposits at the periphery of the lesion (Fig. 5) as well as complete closure of the lesion (Fig. 6). Necrosis and excessive cell debris representing the chronic stage of the disorders (Fig. 7) occurred in diseased cod from the Gdańsk Bay with a prevalence equal to only 4% (Fig. 3). Skin disorders in the cod appeared to be the least prevalent in the northern part of the Bornholm Basin area. They were estimated at p = 7% and p = 2.5% in 2016 and 2017, accordingly (Fig. 2). Comprehensive data on the prevalence of skin disorders in the Baltic cod broken down by fishing area is presented in Figs 2 and 3.
Fig. 2.
Prevalence of Baltic cod ulceration by fishing ground and International Council for the Exploration of the Sea Sub-divisions
Fig. 3.
Stages of Baltic cod ulceration in 2016 and 2017 by fishing ground
Fig. 4.
Skin ulceration observed in a Baltic cod – acute and first-stage inflammatory process with visible flat erosions of the skin
Fig. 5.
Skin ulceration observed in a Baltic cod – beginning of the healing process with scar formation and melanin deposits
Fig. 6.
Skin ulceration observed in a Baltic cod –final stage of the course of ulceration with healing and star-shaped closure
Fig. 7.
Skin ulceration observed in Baltic cod – chronic stage of the course of ulceration. a, b – advanced disease with tissue swelling, developed inflammation and tissue lysis; c, d – advanced disease process with tissue necrosis
Among the Baltic cod caught in the Gdańsk Bay fishing ground, 1% exhibited deep necrotic lesions defined by their regular round shape (Fig. 8). Lesions at the other extreme of severity, which were flat, irregular, shallow and superficial, were observed only in fish from the Bornholm Basin area (Fig. 9).
Fig. 8.
Skin ulceration observed in a Baltic cod – chronic stage of the course of ulceration: advanced disease process with developed inflammation, tissue lysis and deep necrotic lesions
Fig. 9.
Skin ulceration observed in a Baltic cod – flat, irregular, superficial skin lesions without tissue inflammation
More than 1,200 fish were clinically healthy. Parasitological examinations of the lesions on these fish did not reveal the presence of parasites.
Approximately 850 bacterial isolates originating from Baltic cod skin disorders were collected and identified during our study. Based on the analysis of their biochemical properties with the API and VITEK system tests, they were classified to genus and species levels. Among the collected strains, groups of bacteria such as the
The bacterial species identified from skin ulcers differed by the fishing ground where the cod was caught (Table 1). The microorganisms predominating in the Polish EEZ represented different species:
Groups of bacteria isolated from skin ulcers of Baltic cod in correlation with the fishing area
| Fishing area | Bacteria |
|---|---|
| Gdańsk Bay | |
| Słupsk Furrow | |
| Kołobrzeg-Darłowo | |
| Bornholm North | |
| Bornholm South |
Phylogenetic analysis based on the 16S rRNA was performed on the PCR products of 145 bacterial strains representing the two most numerous groups of microorganisms, biochemically identified as the
Fig. 10.
Analyses of the 16S rRNA gene of
Molecular analysis of the PCR products of 72 isolates belonging to the
Fig. 11.
Analyses of the 16S rRNA gene of
The phenotypical characteristics of the isolated bacteria which were investigated included their antimicrobial resistance. The MICs are epidemiological cut-off values categorising isolates as possessing (NWT) or not possessing (WT) mechanisms that reduce their susceptibility to drugs. They were determined for the chosen groups of bacteria and are presented in Fig. 12. Wild-type and NWT isolates were distinguished in each tested group of microorganisms. The highest number of NWT strains (≥8) was found with resistance against sulphamethoxazole and amoxicillin and clavulanic acid in all groups of studied isolates. The diversity of WT and NWT distribution among individual bacterial groups was evident in the collected isolates of
Fig. 12.
Minimal inhibitory concentration (MIC) values determined for bacteria collected from the ulcers of Baltic cod. CIP – ciprofloxacin; ENRO – enrofloxacin; FLUQ – flumequine; AUG2 – amoxicillin and clavulanic acid; FFN – florfenicol; DOX – doxycycline; OXY – oxytetracycline; SMX – sulphamethoxazole
Numerous studies regarding fish health disorders, including Baltic cod skin ulceration, have been conducted since the 1980s (27, 28, 29). During this period, various trends in the extent of the diseases in cod were recognised (29, 40). For example, the highest prevalence of fish ulceration (40%) was recorded in 1982 in the Bornholm area (28), while in 1990, only 5% of fish from there were observed to be diseased (36). At the beginning of the 2000s, the prevalence of Baltic cod skin ulceration increased in the southeastern part of the Baltic Sea (23, 24), and in this location, mainly in the Gdańsk Bay as one of the species’ primary spawning areas, increased episodes of skin ulceration in Baltic cod have been recorded regularly since the beginning of the 21st century (7, 23, 24). Our studies confirmed these data, indicating the Gdańsk Bay as an area with a higher number of diseased cod than any of the other four research fishing grounds.
The clinical signs observed in cod were described as an externally visible fish disease (EVFD), a term defining pathological changes commonly found in wild marine fish and used as indicators in environmental monitoring programmes (41). These pathological changes may have an infectious (
During our study of Baltic cod, various stages of development were detected in the visible skin disorders (Figs 4–9). As mentioned above, the observed pathological signs of this kind may have an infectious aetiology. The pertinent literature offers limited data from comprehensive microbiological studies of such skin lesions in Baltic cod. Mallergaard and Bagge (42) described the isolation of
Infections caused by motile mesophilic
Diversity among the
The research has shown that which bacteria could be isolated from Baltic cod skin ulcers depended on the fishing area. Regardless of the region,
Epidemiological cut-off values estimated by MIC aim to categorise isolates based on whether they possess mechanisms that reduce their susceptibility to antimicrobials (53). However, interpreting the obtained results is difficult because the available data with their interpretative criteria are limited (54). Considering the global threat of the spread of bacterial resistance to antimicrobials, the presence of
The occurrence of health disorders in fish may relate to the pollution of the Baltic Sea. Most parts of this area were classified as “disturbed by hazardous substances”, the substances being heavy metals and persistent organic pollutants but also dumped chemical munitions, including chemical warfare agents from the Second World War (26, 27, 29). Those substances can irritate the skin through direct contact, causing damage and erosion. The evidence of the effect of such hazardous substances could be noted in the Baltic cod caught around the Bornholm area, where around 34,000 tonnes of chemical munitions, including approximately 12,000 tonnes of chemical warfare agents, were dumped east of Bornholm and near Gotland in 1947 (27). Our studies indicated atypical skin lesions in fish caught near the Bornholm area (Fig. 9), which differed from those found in cod caught in other regions of the Baltic Sea (Figs 4–7). These disorders were not characteristic of the clinical picture of the typical ulcers caused by bacteria. The skin lesions were shallow and did not penetrate deep into the tissues in these cases. There was no zone of inflammation and necrosis characteristic of the ongoing process resulting from the infection caused by the bacteria. Therefore, the theory regarding the impact of exposure to chemical substances in the Baltic Sea on fish health remains unproven. The mechanism of the effect of the chemicals and other contaminants on fish skin is insufficiently studied. It is known that sustained exposure to even low concentrations of anthropogenic pollutants can injure the epithelium of fish and cause immunosuppression (4). On the other hand, other unusual skin lesions in regular circular shapes have been observed in some Baltic cod caught in the Gdańsk Bay (Fig. 8). Such clinical signs, similar in shape to the mouth of a lamprey, in combination with the location where the fish bearing these injuries were caught, may suggest that the fish were bitten by European river lamprey (
It was shown that different bacteria species could be isolated from Baltic cod skin ulcers. Most of them were indicated to be opportunistic fish pathogens, which are known to be widespread in the aquatic environment and potentially harmful to humans.