Cent. Eur. J. Biol. • 7(5) • 2012 • 878-885 DOI: 10.2478/s11535-012-0066-8

Central European Journal of Biology

Aeromonas salmonicida infected fish transfer disease to healthy fishvia water

Research Article Vesta Skrodenytė-Arbačiauskienė*, Nijolė Kazlauskienė, Milda Zita Vosylienė, Tomas Virbickas

Institute of Ecology, Nature Research Centre, LT-08412 Vilnius, Lithuania

Received 23 January 2012; Accepted 25 May 2012

Abstract: Experimental studies of infection transmission via water from infected to healthy fish were conducted. The dark-brown bacterial colonies typical for Aeromonas salmonicida on tryptone soya agar (TSA) have been isolated and counted (from 3.0±0.6x102 to 3.5±0.5x105 c.f.u. g-1) from the internal organs of naturally infected (NI) and experimentally infected (EI) perch and sea trout. No significant differences in dark-brown bacterial counts were detected between EI perch and EI sea trout. The assessment and comparison of the alterations of the biological parameters of EI European perch and sea trout with bacterium Aeromonas salmonicida subsp. salmonicida with naturally infected perch were conducted. No mortality was recorded in groups of EI perch and sea trout. Whereas, the mortality of NI perch (collected from the main sites of outbreak of disease) was observed from the second day of the experiments. Changes in morphophysiological parameters of EI perch and sea trout were similar. Different alterations in blood cell parameters of EI fish were observed, and the most noticeable was the decrease (P≤0.01) in white blood cell count (WBC) of EI perch and sea trout. Based on these results it can be deduced that there is infection transmission of bacterium A. salmonicida from European perch via water to other fish species.

Keywords: Aeromonas salmonicida subsp. salmonicida • Infection • Transfer • European perch • Sea trout © Versita Sp. z o.o.

1. Introduction water bodies might cause severe declines in wild fish populations; however, main triggers inducing the disease Mass mortalities in European perch (Perca fluviatilis L.) outbreak and the high mortality remain unknown. occurred in North Lithuanian Rivers (Lėvuo, Mūša and The present investigation of the experimental Nevėžis) in 2008 and reached a maximum in mid-autumn infection is a continuation of our previous study [1], at a water temperature of 6ºC. Pathogenic Aeromonas and referred to the transmission and impact of infection salmonicida subsp. salmonicida were identified as a with Aeromonas salmonicida subsp. salmonicida of possible causative agent of fish disease - septicaemia, European perch and sea trout. Infection transmission which usually resulted in high mortality rates [1]. The to healthy perch and sea trout in this study was bacterial isolates were identified by sequence analysis of conducted via water from infected perch collected in the 16S rRNA gene [1]. Whilst European perch (Perca the known main site of disease outbreak. Numerous fluviatilis L.) are commonly widespread in European experimental studies have examined the transmission water bodies, data available on this disease in wild perch of the A. salmonicida via water by bath immersion [3], or are scarce. Epizootic of sub-acute furunculosis caused intraperitoneal and intramuscular injections at different by Aeromonas salmonicida was described among perch bacterial doses of different farm-reared fish [4-7]. in a drainage channel in 1972 [2]. A similar accident However, until this study, there were no references to was followed by high mortality of perch as recorded in infection transmission via water from infected wild fish West Ukraine, Dnestr River, and Dnestr water reservoir to healthy fish of different species. Laboratory-controlled in October-November 2010 (Khudyi O., personal investigation of the transmission of infection from communications). Outbreaks of disease in natural wild fish should provide new information towards the

* E-mail: [email protected] 878 V. Skrodenytė-Arbačiauskienė et al.

validation of disease occurrence in wild and farm-reared unpublished data, 2008). No parasitic and viral infections fish and the general health condition of infected fish of were detected in experimental fish (Report of Lithuanian different species. National Food and Veterinary Risk Assessment Institute, Gills, skin, and wounds are likely to be the main http://www.Balsas.lt/naujiena/222156). routes of entry for A. salmonicida [6,8]. It has been shown All tests were performed using artesian water of high that particulate bacterial and viral agents are readily quality. Average hardness of water was ~284 mg/l as - absorbed via the gills [9]. Bacteria can also enter the host CaCO3, alkalinity was 244 mg/l as HCO3 , mean pH was by the fins [10], and can cross the gastrointestinal lining in 8.0, temperature was ~6-8oC, and oxygen concentration three different ways. In undamaged tissue, bacteria can ranged from 8 to10 mg/l. translocate by transcellular or paracellular routes [11]. The approval of the animal use in the experiments The presence of infection can be diagnosed via changes was given by the Ministry of Environment of Lithuania in the morphophysiological, physiological, microbiological 2009-04-10 No 9F09-21. parameters of the fish. Quantitative aspects such as survival, growth, weight-length relationship, condition 2.2 Experimental design factor, and tissue-somatic indices of fishes are important Experimental procedures are shown schematically tools in the study of fish biology [12]. The condition factor (Figure 1). After the acclimation period experimental reflects information on the physiological status of fish fish (EI) were transferred from holding tanks and [13]. Haematological parameters (red and white blood placed into 80-l constantly aerated aquaria. The first indices) and microbiological (bacterial colonies count) group of healthy perch were placed into two aquaria endpoints are indicative in diagnosing and evaluating the (aquarium 1, 2), which served as controls (N=20). functional status of fish 14[ ,15]. They are considered to Infection assays were conducted using the second group be a reliable approach in the assessment of the affect of of control perch placed into two other aquaria (aquarium pathogens on fish [9]. 3, 4) (N=14) (experimentally infected – EI) to which the The aim of the present study was to investigate group of marked perch collected from the main site of experimentally if Aeromonas salmonicida infections can disease outbreak (naturally infected – NI) (N=14) were be transferred via water from infected European perch to transferred. The third group of fish (two aquaria – 5, 6) wild healthy perch and to healthy fish of different species consisted of the perch collected from the main site of (sea trout); to evaluate the alterations in the biological disease outbreak (N=20). The fourth group – healthy sea (microbiological, morphophysiological, physiological) trout was kept in two aquaria (aquarium 7, 8) as controls parameters of experimentally infected perch and sea (N=20). The fifth group of control sea trout was also placed trout, and to compare with the same indices of healthy into two aquaria (experimentally infected – EI) (N=14) perch and sea trout and of naturally infected perch. and infected perch (naturally infected – NI) (aquarium 9, 10) were gently transferred to these aquaria. Fish were fed a dry commercial feed (DANA-FEED, Denmark) daily. 2. Experimental Procedures All fish were sampled during days 5-8 taking both control and infected fish on each day. 2.1 Fish sampling Live perch (Perca fluviatilis L.) (naturally infected 2.3 Morphological, morphophysiological and – NI group) were collected from the Nevėžis River (the haematological examination main site of disease outbreak and of the high mortality The physiological state of the fish was assessed by use of the perch), and from the Savenes River outflowing of biological parameters. Morphological parameters from Kazimieravo Lake, where no mortality of perch was studied were: mean body weight (Q) 29.62±2.0 g and recorded (healthy, control fish group). Fish were caught mean length (L) 14.5±0.3 cm of control, 29.54±2.2 g using gill nets with a mesh size 12 mm and transported and 14.2±0.4 cm of NI, 32.82±3.0 g and 14.3±0.4 of in aerated tanks to the Institute of Ecology of Nature EI (M±SEM) of perch; 11.0±2.6 g and 10.8±0.2 cm of Research Centre (Vilnius) and kept alive in aerated control, 12.6±2.0 g and 10.2±0.4 cm EI (M±SEM) of aquaria until examination. Sea trout (Salmo trutta trutta) sea trout. Weight of spleen, gills, liver, heart (g), and were reared in laboratory and kept separately in aquaria. weight of fish without viscera (g) were measured. Sea trout had no history of A. salmonicida infection and Morphophysiological parameters evaluated including A. salmonicida bacteria were not found in liver and the: condition factor (CF) and tissue-somatic index kidney of healthy, control perch. No pollutants in the (TSI). CF was calculated using the formula [13]: water of Nevėžis and Savenes Rivers were determined Condition factor = [the weight of the fish (g) x the (Report of Hydrometeorology Service of Lithuania, length of fish3 (cm)]x100%.

879 Aeromonas salmonicida infected fish transfer disease to healthy fishvia water

Figure 1. The experimental procedure scheme.

Spleen-somatic (SSI), gill-somatic (GSI), liver- weighted and homogenized in a ninefold volume of a somatic (LSI), heart-somatic (HSI) indices were diluent of phosphate-buffer (PB; 10 mmol l-1 phosphate

calculated using the formula [16]: (Na2HPO4), pH 7.2). Each sample was serially diluted Tissue-somatic index = [tissue weight (g) x¸ weight and 0.05 ml portions of diluents were spread over of fish without viscera (g)]x100%. duplicate plates on tryptone soya agar (TSA, Oxoid) For each fish, the erythrocyte count (RBC, for detection A. salmonicida. Occurrence of colonies 106 mm-3), the haemoglobin concentration (Hb, g l-1), surrounded by dark-brown water soluble pigment on the haematocrit level (Hct, l l-1) and the white blood cell TSA after incubation at 22ºC for 3 days, is considered count (WBC, 103 mm-3) were determined using routine indicative of the presence of A. salmonicida and these methods [17]. colonies were counted (colony forming units, c.f.u.) [18]. Afterwards, dark-brown colonies were selected 2.4 Bacteriological investigation and restreaked three times onto fresh media to obtain Naturally infected (NI) perch (N=20), control perch pure isolates. A. salmonicida was identified by DNA (N=6) and control sea trout (N=6) were bacteriologically extraction, REP-PCR, amplification and sequencing of investigated before the experiment. For bacteriological 16S rRNA gene [1]. After 8 days incubation count of analysis, sterile instruments were used to collect dark-brown colonies on TSA from tested fish have been samples from the liver and kidney of fish. Samples were repeated.

880 V. Skrodenytė-Arbačiauskienė et al.

2.5 Statistical analysis of EI perch. The elevation of bacterial counts in EI perch The data obtained were expressed as mean ± standard organs was similar to those observed in the same error of the mean (M±SEM) and as mean±standard organs of NI fish. A similar elevation in the dark brown deviation of the mean (M±SD). Significance of all data bacterial count in organs of EI sea trout was observed obtained was verified by Student‘s t-test at P≤0.001, with the greatest increase (2.0±0.3x105 c.f.u. g-1) P≤0.01, P≤0.05, using the programme GraphPAD InStat in liver of fish. The dark brown bacterial counts in the (USA). liver of EI perch and sea trout were very similar, while this parameter in the kidneys of sea trout consisted of 3.0±0.6x102 (c.f.u. g-1) and was significantly increased 3. Results as compared to that in perch kidneys (Table 1). No significant changes were found in CF, LSI and The mortality of NI perch (collected from the main HSI of EI fishes as compared with the same parameters sites disease outbreak) was recorded in the 3-rd group of control perch (Table 2), whereas the CF and LSI from the second day (a total 30% of fish died). Visual values of EI and NI perch tended to increase. Significant examination at the end of exposure showed that 86% of decrease in GSI (P<0.01) and SSI (P<0.01) of EI perch EI fish had lesions at the base of caudal and dorsal fins. was detected. Significant differences were also found in No A. salmonicida bacteria were found in the liver morphophysiological parameters of control and EI sea or kidneys before the experiment and after 8 days of trout (Table 2). In contrast to EI perch, CF and LSI of incubation in the control perch and sea trout (Table 1). EI sea trout were significantly (P≤0.01 and P≤0.0001, The values of viable bacterial (dark brown) counts typical respectively) increased, while GSI, and HSI markedly for A. salmonicida on TSA [18] from the same tissues (P≤0.01) decreased as compared to controls. of EI perch highly increased after 8 days of incubation The erythrocyte count, haemoglobin concentration and were significantly higher as compared to the and WBC count of EI perch was higher (not significantly) controls. The most dramatic changes were registered or significantly increased as compared to indices of in liver bacterial counts (2.4±0.5x105) (c.f.u. g-1) control fish (Table3 ). Hct level remained at a level similar

Liver Kidney Groups (c.f.u./g) (c.f.u./g)

1-st, control perch (N=20) 0±0 0±0 2-nd, experimentally infected perch (EI) (N=14) 2.4±0.5x105 8.0±1.0x102

3-rd, naturally infected (NI) perch (N=20) 3.5±0.5x105 1.9±0.2x103

4-th, control sea trout (N=20) 0±0 0±0

5-th, experimentally infected sea trout (EI) (N=14) 2.0±0.3x105 3.0±0.6x102

Table 1. Bacterial counts (dark - brown) on TSA in the liver and kidney of control, experimentally infected (EI) and naturally infected (NI) groups of European perch Perca fluviatilis and sea trout Salmo trutta trutta (M±SD) (after 8 days incubation).

Notes: * standard deviation

Groups CF LSI GSI SSI HSI

European perch

1-st, control (N=20) 0.97±0.1 1.79±0.12 5.07±0.12 0.22±0.03 0.14±0.01

2-nd, experimentally infected (EI) (N=14) 1.03±0.1 1.81±0.16 4.65±0.10* 0.19±0.02* 0.13±0.01

3-rd, naturally infected (NI) (N=20) 1.13±0.03* 1.86±0.12* 4.80±0.10* 0.19±0.03* 0.13±0.01

Sea trout

4-th, control (N=20) 0.87±0.03 0.94±0.12 4.39±0.12 0.17±0.03 0.21±0.01

5-th, experimentally infected (EI) (N=14) 1.19±0.04* 1.81 ± 0.16* 3.93±0.15* 0.13±0.02 0.15±0.02*

Table 2. Morphophysiological parameters of control, experimentally infected (EI) and naturally infected (NI) groups of European perch Perca fluviatilisand sea trout Salmo trutta trutta (M±SEM).

Notes: * values significantly different from controls (P≤0.0001).

881 Aeromonas salmonicida infected fish transfer disease to healthy fishvia water

Er, Hb, Hct, WBC, Groups 106 mm-3 g/l l/l- 103 mm-3

European perch

1-st, control (N=20) 1.29±0.08 79.9±5.7 0.37±0.02 35.2±3.6

2-nd, experimentally infected (EI) (N=14) 1.83±0.1* 84.7±7.5 0.37±0.01 85.9±14.4**

3-rd, naturally infected (NI) (N=20) 1.73±0.09** 79.7±7.1 0.36±0.02 16.3± 2.8**

Sea trout

4-th, control (N=20) 0.87±0.07 85.9±2.7 0.33±0.01 21.8±3.8

5-th, experimentally infected (EI) (N=14) 1.0±0.03 77.3±5.5 0.28±0.01* 45.4±5.9**

Table 3. Haematological parameters of control, experimentally infected (EI) and naturally infected (NI) groups of European perch Perca fluviatilis and sea trout Salmo trutta trutta (M±SEM).

Notes: * values significantly different from control group values (P≤0.01, 0.02), **values significantly different from control group values (P≤0.0001).

to the control in all three groups studied. Whereas WBC in which no mortality of EI perch and sea trout was count in EI perch was significantly elevated (P≤0.0001), observed during the experiment period, but higher and in contrast, WBC in blood of NI perch was found viable bacterial counts were isolated in the organs to be decreased. The erythrocyte count in the blood of (liver and kidney) of EI fish. The acute forms caused EI sea trout was not significantly higher, as haemoglobin high mortality (observed among the fish collected from concentration, while Hct level was significantly lower the sites of disease outbreaks) while in subacute and (P≤0.01) as compared to controls. The WBC count was chronic forms the onset of the disease is more gradual highly elevated (P≤0.0001). This increase was found in and mortality relatively low [10]. The study of the humoral the blood of 75% of trout studied, however, the reduction response of Atlantic salmon (Salmon salar L.) showed in WBC count to 13.8 103 mm-3 of cells in the blood of the differences in responses between chronically and some trout was recorded. recently infected salmon, when the recently infected fish produced greater responses [20]. The morphophysiological parameters of EI perch 4. Discussion and sea trout changed in a similar pattern depending on fish species and tissues examined. The greatest The death (30%) of perch collected from the sites alterations were found for sea trout, e.g. CF and LSI of disease outbreak was observed from the second of EI perch tended to increase, while the significant day of placement into the aquaria, whilst no mortality increase of these parameters was found in EI sea was recorded in any of the groups of EI perch and trout. In contrast, GSI and SSI of EI perch and GSI of sea trout kept with NI perch during the experiment. It EI sea trout significantly decreased (P≤0.01). These was reported by other authors that fish infected with data, in general, are consistent with McCarthy [2] that A. salmonicida subsp. salmonicida that died from this fish of the family Salmonidae are considered to be the bacterium released toxins, which induced the necrosis most susceptible to furunculosis, particularly brook trout of parenchymatous tissues of fish as liver, spleen, and (Salvelinus fontinalis (Mitchill)), Atlantic salmon (Salmo kidney, and caused necrotic lesions in gills, gut and salar L.) and also brown trout (Salmo trutta L.), with muscle [10,19] and (Moeller R. B. Jr., DVM, Bacterial rainbow trout (Oncorhynchus mykiss (Walbaum)) being Diseases of Fish, California Animal Health and Food, relatively resistant. According to Australian scientists http://www.aquaworldnet.com/awmag/diseases. furunculosis may affect all species and all ages of both htm#sense). freshwater and marine salmonids, with brook trout The typical A.salmonicida dark brown bacterial (Salvelinus fontinalis) and brown trout (Salmo trutta) colonies on TSA have been isolated and counted (from being particularly susceptible. It is emphasized (Disease 3.0±0.6x102 to 3.5±0.5x105 c.f.u. g-1) from the internal strategy, Infectious salmon anaemia, Version 1.0, 2009, organs of NI and EI perch and sea trout. No significant http://www.daff.gov.au/animal.../1_nature_of_the_disease) differences in A.salmonicida (c.f.u. g-1) were detected that although it is likely that the greatest impact of the between EI perch and sea trout. It is deduced that disease will be on salmonid fish, non-salmonid fish according to Hiney et al. [10] “clinically inapparent” or species are capable of being infected and may transfer “covert” forms of infection were observed in our study the pathogen.

882 V. Skrodenytė-Arbačiauskienė et al.

Different alterations in red blood cell parameters pathogen multiplication and host defence mechanisms were found in EI perch and sea trout. Significant is an important relationship [30]. increases in erythrocyte counts in EI perch and slight Based on the experimental results it was deduced elevation in erythrocyte counts in sea trout contradict that healthy perch and sea trout were infected from the findings by Harikrichnan et al. and Rehulka [21,22], naturally infected perch during the 5-8 days period. which reported the decrease in erythrocyte counts of This is confirmed by scientific data that Aeromonas common carp infected with A. hydrophila and rainbow bacteria induced diseases, which are transmitted trout infected with Aeromonas strain. By contrast, through contaminated water or contacted with haemoglobin and haematocrit levels did not change infected fish (Moeller R. B. Jr., DVM, Bacterial in EI perch and tended to decrease or significantly Diseases of Fish, California Animal Health and Food, decrease in EI sea trout. Similar decreases in these http://www.aquaworldnet.com/awmag/diseases.htm#sense). parameters were observed in Aeromonas-infected carp Our research has shown that sea trout were infected [21,23] and Aeromonas strain infected rainbow trout in a similar manner to the control perch from infected [22]. Decreases in haematocrit levels were also found perch. in Atlantic salmon infected with A. salmonicida [24], and eels spontaneously infected with A. hydrofila [25]. Considerable elevation of WBC count in blood of EI 5. Concluding Remarks perch and sea trout was found. A similar increase in WBC count was reported in carp infected with A. hydrophila Our experimental study proved that the infection with [21]. It is deduced that this increase in the WBC count bacteria Aeromonas salmonicida subsp. salmonicida could be explained by the initial stages of infection in EI transmission via water from NI European perch perch and EI sea trout. However, the reduction in WBC (collected from the main site of disease outbreak) to count was observed in Aeromonas-infected carp from healthy wild perch and artificially bred sea trout occurred. the first day of infection and remained low until day 8 This deduction was confirmed by alterations obtained [23]. Ulcerative A. salmonicida infections occured in carp in the biological (microbiological, morphophysiological, in addition to induced leukopenia and a state of immune physiological) parameters of EI perch and sea trout. The suppression [26]. Similar suppression of the immune bacterial count (c.f.u. g-1) in the internal organs of NI and system (immunoglobulin significantly decreased) in carp EI perch and sea trout was detected to be higher than infected with A. salmonicida was found by Pourreau that of organs (liver, kidney) of non-infected fish. No et al. [27]. However, the modulation of immunological significant differences in bacterial colonies units were parameters was reported in brook trout (Salvelinus detected between EI perch and sea trout. Comparison fontinalis) infected with A. salmonicida [28]. The different of morphophysiological parameters of EI perch and sea alterations in haematological parameters of EI perch trout showed a similar trend of obtained changes. While and EI sea trout observed in this study are in agreement greater alterations in haematological parameters of with previous results of Dhanaraj et al. [29] for four fish EI sea trout than in EI perch were found. Based on these species infected with A. hydrophila. According to these results, it was deduced that the infectzion transmission of authors, the different dynamic changes in values of red bacterium Aeromonas salmonicida subsp. salmonicida and white blood cell parameters for different species of via water to other fish species occurred. Changes fish were recorded. in environmental conditions (water temperature, pH, Our data demonstrated greater alterations in oxygen content in water), the deterioration of fish haematological parameters of EI sea trout then in EI perch. physiological state stimulate reproduction and the Furthermore, the differences in immune responses of spread of Aeromonas salmonicida subsp. salmonicida infected fish could depend on their physiological state infection via water bodies. This is very hazardous to wild and disease duration [29]. Similar different alterations in and farmed fish species. WBC counts were found in our previous study with perch naturally infected with A. salmonicida [1]. Scientific data showed that at 14°C, the period Acknowledgements from exposure of healthy fish to A. salmonicida subsp. salmonicida (by cohabitation with infected fish) to This work was funded by the Ministry of Environment bacterial shedding can be as short as three days [3]. of Lithuania grant 2009-04-10 No 9F09-21. The authors Whilst at lower temperatures (6-8°C in case of our would like to thank M. Richardson, BASIC, Crawley, studies) the time between infection and death could England for reading, commenting on and improving the be prolonged (5-8 days). The effects of temperature on English of this article.

883 Aeromonas salmonicida infected fish transfer disease to healthy fishvia water

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