Effects of Water Temperature on the Susceptibility of Rainbow Trout to Streptococcus agalactiae

Transcription

1 Acta Scientiae Veterinariae, : 097. RESEARCH ARTICLE Pub.097 ISSN Effects of Water Temperature on the Susceptibility of Rainbow Trout to Streptococcus agalactiae Ali Sepahi,2, Marzieh Heidarieh 2, Alireza Mirvaghefi, Gholam Reza Rafiee, Mehrnoush Farid & Najmeh Sheikhzadeh 3 ABSTRACT Background: The outbreak of diseases is a limiting factor in fish culture. Among different bacterial agents, Streptococcus agalactiae is a major problem, causing heavy loss in cultured and wild fish species from fresh and marine water. Outbreaks with considerable mortalities in different fish species have been associated with multiple environmental factors, including warm water temperatures, increased ammonia levels and low dissolved oxygen levels. The effects of various water quality factors on streptococcal infections have been previously shown. To date, there is no available data on the effects of water temperature on the infection of rainbow trout with S. agalactiae. Therefore, present study evaluated the effect of two water temperatures (2 and 8ºC) on clinical signs, relative percent survival (RPS), cumulative mortality and LD50 in rainbow trout against S. agalactiae. Materials, Methods & Results: Three hundred and sixty rainbow trout (56 ± 4. g) were acclimatized for 2 weeks and fed the commercial fish diet at 2% body weight twice daily. The experiment was conducted in two different water temperatures (2 ± ºC and 8 ± ºC). In each temperature, fish were injected with 0.2 ml of five different concentrations (0 8, 0 7, 0 6, 0 5 and 0 4 CFU ml - ) of S. agalactiae (RTCC205). In control group, the same amount of physiological saline was injected instead of the bacterial suspension. Mortality rates and clinical signs were recorded for 4 days. Throughout the experiment, different signs such as eye abnormalities, erratic swimming and body lesions were observed in fish which were exposed to high bacterial doses at 8ºC while fish kept at 2ºC were completely asymptomatic. No mortality was observed within 2 or 3 days in fish at 2ºC while at 8ºC, the mortality started growing rapidly even from first day of bacterial injections. Results also revealed that the trend of mortalities in 8ºC was quite different from 2ºC. The median lethal dose (LD50) on the 4th day was also calculated at 2 and 8ºC as CFU ml - and CFU ml -, respectively. Discussion: In this study, the relationship between temperature and infection with Streptococcus agalactiae was pronounced. When the water temperature was 8ºC, mortality due to S. agalactiae was higher in rainbow trout than 2ºC. It was previously showed that the optimum temperature for growth provide the best performance of fish immune system. Non-specific and specific immune parameters in teleost decrease significantly when the water temperature is lower or higher than normal physiological range of the fish. According to current study, the higher mortality in fish kept at 8ºC might be affected by the reduction of rainbow trout immunogenic activity. On the other hand, the effects of water temperature on the progress of infection in fish species are also important. For example, manipulation of temperature and/or salinity can be used to reduce the growth rate of Flavobacterium psychrophilum. The change of virulent S. agalactiae related to water temperature in fish species needs more consideration. In conclusion, there was a clear relationship between temperature and mortality in rainbow trout against Streptococcus agalactiae and the total cumulative mortality at 8ºC was higher compared to 2ºC for every single concentration used. Keywords: water temperature, Streptococcus agalactiae, LD50, rainbow trout, relative percent survival. Received: 5 March 202 Accepted: 3 September 202 Published: 3 January 203 Department of Fishery and Environment, Faculty of Natural Resources, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran. 2 Agricultural, Medical and Industrial Research School (AMIRS-NSTRI), Karaj, Iran. 3 Department of Food Hygiene and Aquatic Animals, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran. CORRESPONDENCE: M. Heidarieh [mheidarieh@nrcam.org - Fax: +98 (26) 440]. Agricultural, Medical and Industrial Research School (AMIRS-NSTRI). P.O.Box: Karaj, Iran.

2 INTRODUCTION Fish in production facilities are exposed to stressful conditions, diseases and deteriorating environmental conditions, that can have serious economic losses [2]. Among different bacterial agents, Streptococcus agalactiae is the cause of streptococcosis in numerous fish species, including tilapia, Oreochromis niloticus (L.), and mullet, Liza klunzingeri [6], golden shiners, Notemigonus crysoleucas [3], menhaden, Brevoortia patronus [0], and bullminnows, Fundulus grandis []. The pathogen can produce septicemia and meningoencephalitis in diseased fish, which show various clinical signs such as skin hemorrhage, exopthalmia, ascites and erratic swimming concerned as a typical sign for streptococcosis [9]. There have been a number of studies examining the effects of various water quality factors such as low oxygen levels and increased nitrite, over crowding on streptococcal infections in fish species [4,5]. For aquatic animals, water temperature also play an important role on the prevalence of diseases in cultured fish. It was shown that there is a strong association between water temperature and increased mortality, especially between 25 and 28ºC in barramundi (Lates calcarifer) [3]. Rodkhum et al. [4] recently showed that Nile tilapia reared in high water temperature is highly susceptible to S. agalactiae. To date, there is no available data on the effects of water temperature on the infection of rainbow trout with S. agalactiae. Therefore, this study was carried out to investigate the effects of two water temperatures (2 and 8 C) on clinical signs, relative percent survival (RPS), cumulative mortality and LD50 in rainbow trout against Streptococcus agalactiae. MATERIALS AND METHODS Conditions of the experiment Healthy rainbow trout with average body weight 56 ± 4. g were obtained from Mahisara fish farm (Karaj, Iran). Study was started from September 200 and specimens were exposed to a natural photoperiod (2L : 2D). They were acclimated in 300 L tanks filled with chlorine-free tap water and provided with continuous aeration using an electric air pumping compressor for 2 weeks. During the experiment, the following conditions were maintained: dissolved oxygen concentration 6.0 ± 0.6 mg L - and ph 7.0 ± % of the water was exchanged twice a week to remove waste feed and fecal materials. Throughout the acclimation period, the fish were fed the commercial fish diet at 2% body weight twice daily and continued two weeks after injections. Bacterial preparations Streptococcus agalactiae (RTCC205) was cultured on brain heart infusion agar (BHI) in 37ºC for 24 h. Cells were harvested, suspended in physiological saline, to obtain a final concentration of 0 8 CFU ml -. From an initial bacterial concentration of 0 8 CFU ml -, 0-fold serial dilutions were prepared (0 8, 0 7, 0 6, 0 5 and 04 CFU ml - ). Experimental infection trial Three hundred and sixty fish were divided into 2 groups with subgroup of 5 different concentrations (T5:0 8, T4:0 7, T3:0 6, T2:0 5 and T:0 4 CFU ml - ) along with the control group in triplicate with 0 fish in each tank. The experiment conducted in two different water temperatures (2 ± and 8 ± ºC). In each experiment, fish were injected with 0.2 ml of each dilution intraperitoneally. In control group, the same amount of physiological saline was injected instead of the bacterial suspension. Mortalities were recorded daily for 4 days. Dead fish were removed from the tanks daily and their livers and kidneys were subjected to bacterial identification testing to confirm the presence of Streptococcus agalactiae and the clinical signs were also reported. The LD50 was calculated and expressed as colony forming units per milliliter (CFU ml - ) [2]. RPS was calculated by the following formula of Ellis [5]: RPS = - { Percent mortality in treated group Percent mortality in control group } x 00 Statistical analysis All statistical analyses were conducted by SPSS. Determination of the LD 50 was conducted using Probit analysis, and mortality schedules were compared using the survival function with pairwise comparison. RESULTS In the current study, rainbow trout challenged with Streptococcus agalactiae at 8ºC exhibited various signs and lesions such as exophthalmia, haemorrhaging at the base of fins, visceral haemorrhage and ascitic fluid accumulation in the peritoneal cavity present in all fish dying from exposure at 8ºC while there was no clinical sign observed in 2ºC (Figure ). 2

3 A Figure. Lesions of rainbow trout challenges with Streptococcus agalactiae via water immersion route. A: Petechial hemorrhage at pyloric cecae (arrow) and enlarged reddened spleen (arrow head), B: Kidney enlargement (arrow) and congestion in pale liver (arrow head), C: Ascites in the peritoneal cavity (arrow), D: Hemorrhage at base of the fins. This study showed that, cumulative mortality at 2ºC was considerably lower than 8ºC in all treatment groups while in each temperature, highest cumulative mortality was occurred in T5 (0 8 ) group. In fish kept at 2ºC, no mortality was shown within 2 or 3 days after injection. On the other hand, in fish held at 8ºC, mortality started growing rapidly from the first day post injection (Figure 2-3). RPS in each water temperature also showed that in each treatment dose, lower survival was related to fish reared at 2ºC in comparison with rainbow trout kept at 8ºC. The LD50 on the 4th day was calculated at 2 and 8ºC as CFU ml - and CFU ml -, respectively (Table ). Figure 2. Cumulative mortality due to Streptococcus agalactiae in rainbow trout kept at 2ºC during 4 days. Figure 3. Cumulative mortality due to Streptococcus agalactiae in rainbow trout kept at 8ºC during 4 days. 3

4 Table. Relative percent survival (RPS) and LD 50 in rainbow trout kept at two different water temperatures (2ºC and 8ºC). Bacterial Dose (CFU ml - ) Water temperature 2ºC 8ºC LD 50 (CFU ml - ) DISCUSSION In this study, fish kept at 8ºC showed various signs such as exophthalmia (which may be bilateral), corneal opacity, melanosis, lethargy, loss of orientation, swimming erratically, dorsal rigidity, vertebral deformity, tachypnoea, anorexia, emaciation, or sudden death with few accompanying signs. Besides, the most consistent findings were intracranial edema, ocular alterations resulting from a severe haemorrhagic panopthalmitis, Hemorrhaging at the base of fins, exophthalmia ( pop-eye ), ascitic fluid in the abdominal cavity and haemorrhaging in/on the internal organs. These sings with bacterial identification testing indicated the experimental infection with Streptococcus agalactiae. Noteworthy, fish kept at 2ºC did not show any clinical sings of Streptococcosis. Moreover, no mortality was observed within 2 or 3 days in fish at 2ºC while at 8ºC, the mortality started growing rapidly even from first day of bacterial injections. In this study, the LD 50, and RPS results also showed that fish which were kept at 2ºC were much more resistant to this bacterial infection in comparison with 8ºC. Likewise, previous studies reported different range of streptococcosis in fish, from to 0 8 CFU for trout, barramundi and tilapia []. Disease progression in fish is somewhat variable and has been shown to be dependent on the virulence of the isolate, the host species affected, route of infection, fish age and other environmental and water quality factors []. In this study, the relationship between temperature and infection with S. agalactiae was pronounced. When the water temperature was 8ºC, mortality due to S. agalactiae was higher in rainbow trout than 2ºC. It was previously showed that the optimum temperature for growth provide the best performance of fish immune system [3]. Non-specific and specific immune parameters in teleost decrease significantly when the water temperature is lower or higher than normal physiological range of the fish. For example, barramundi grow optimally between 27ºC and 36ºC. In lower temperature, decrease in immune responses allows the bacterial pathogens to establish and disease to develop [3]. A study in tilapia demonstrated that in temperatures less or higher that optimum (29ºC to 3ºC), fish challenged with S. iniae showed the high mortality rate [4]. According to current study, the higher mortality in fish kept at 8ºC might be affected by the reduction of rainbow trout immunogenic activity. The effects of water temperature on the progress of infection in fish species have been shown. It was found previously that manipulation of temperature and/or salinity can be used to reduce the growth rate of Flavobacterium psychrophilum. Indeed, maintenance of temperature above 20ºC may significantly reduce mortality, which particularly occurs if salinity is kept above 0 g L - [8]. In human S. agalactiae, increase in transcription of some important virulence factors at 40ºC in comparison with 30ºC was also shown [7]. The change of virulent S. agalactiae related to water temperature in fish species needs more consideration. Overall, it can be concluded that there was a clear relationship between temperature and mortality in rainbow trout against Streptococcus agalactiae and the total cumulative mortality at 8ºC was higher compared to 2ºC for every single concentration used. SOURCE AND MANUFACTURER Razi Vaccine and Serum Institute, Karaj, Iran. 4

5 Acknowledgements. The authors are grateful to Medical and agricultural research center (Karaj) and Department of Fishery and Environment Faculty of Natural Resources College of Agriculture & Natural Resources, University of Tehran, (Karaj). Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES Agnew W. & Barnes A.C Streptococcus iniae: An aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. Veterinary Microbiology. 22(-2): Aly S.M., Ahmed Y.A.G., Ghareeb A.A.A. & Mohamed M.F Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish and Shellfi sh Immunology. 25(-2): Bromage E. & Owens L Environmental factors affecting the susceptibility of barramundi to Streptococcus iniae. Aquaculture. 290(3-4): Bunch E. & Bejerano I The effect of environmental factors on the susceptibility of hybrid tilapia Oreochromis nilotcus x Oreochromis aureus to streptococcosis. Israelian Journal of Aquaculture. 49(2): Ellis A.E General principles of fish vaccination. In: Ellis A.E. (Ed). Fish vaccination. London: Academic Press, pp Evans J.J., Klesius P.H., Gilbert P.M., Shoemaker C.A., Al Sarawi M.A., Landsberg J., Duremdez R., Al Marzouk A. & Al Zenki S Characterization of β-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait. Journal of Fish Disease. 25(9): Mereghetti L., Sitkiewicz I., Green N.M. & Musser J.M Remodeling of the Streptococcus agalactiae transcriptome in response to growth temperature. PLoS One. 3(7): e Nematollahi A., Decostere A., Pasmans F. & Haesebrouck F Flavobacterium psychrophilum infections in salmonid fish. Journal of Fish Diseases. 26(0): Noga E.J Fish disease: Diagnosis and treatment. Ames: Mosby electronic Publishing, pp Plumb J.A., Schachte J.H., Gaines J.L. Peltier W. & Carroll B Streptococcus sp. from marine fishes along the Alabama and Northwest Florida coast of the Gulf of Mexico. Transactions of American Fisheries Society. 03(2): Rasheed V. & Plumb J.A Pathogenicity of a non-haemolytic group B Streptococcus sp. in gulf killifish, Fundulus grandis Baird & Girard. Aquaculture. 37(2): Reed L. & Muench H A simple method of estimation of fifty percent endpoints. American Journal of Hygiene. 27(3): Robinson J.A. & Meyer F.P Streptococcal fish pathogen. Journal of Bacteriology. 92(2): Rodkhum C., Kayansamruaj P. & Pirarat N. 20. Effect of water temperature on susceptibility to Streptococcus agalactiae serotype Ia infection in Nile Tilapia (Oreochromis niloticus). Thailand Journal of Veterinary Medicine. 4(3): Stoffregen D., Backman S., Perham R., Bowser P. & Babish J Initial disease report of Streptococcus iniae infection in hybrid striped (sunshine) bass and successful therapeutic intervention with the fluoroquinolone antibacterial enrofloxacin. Journal of World Aquaculture Society. 27(4):